Methods for measuring virulence in soybean cyst nematode

ABSTRACT

Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s) that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Described herein is the discovery of three single nucleotide polymorphisms (SNPs) that reproducibly show allele imbalances between soybean cyst nematode (SCN) grown on resistant and susceptible soybean ( Heterodera glycines ) plants. Two candidate SCN virulence genes, biotin synthase (HgBioB) and a bacterial-like protein containing a putative SNARE domain (HgSLP-1), were tightly linked to the SNPs. Methods, kits, and compositions are provided for using these discoveries to detect and quantify SCN virulence in field samples. Also provided are methods for planting fields in accordance with the results of detecting (or not detecting) virulent SCN in the fields.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the earlier filing date of U.S. Provisional Applications No. 62/078,570 filed Nov. 12, 2014, 62/094,367 filed Dec. 19, 2014, and 62/239,046 filed Oct. 8, 2015. The disclosure of each of these prior applications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter of this disclosure was made with government support under contract number 2009-35302-05315 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

FIELD

This disclosure relates to methods for the identification of soybean cyst nematode virulence.

BACKGROUND

Soybeans are a major cash crop and investment commodity, in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production.

The soybean cyst nematode (SCN), Heterodera glycines, is one of soybean's (Glycine max) most damaging pathogens. In the United States alone, SCN is estimated to cause over one billion dollars in annual soybean yield loss (Wrather & Koenning, J Nematol 38: 173-180, 2006). SCN is an obligate root parasite of soybean and forms an intricate association with it host in order to reproduce. The second-stage juvenile (J2) nematode burrows into the root using its hollow, protrudable stylet to physically and enzymatically disrupt the plant cells (Smant et al., Proc Natl Acad Sci USA 95: 4906-4911, 1998). Once the nematode reaches the vascular cells of the root it injects a complex mixture of proteins into a vascular cell, via its stylet, causing the formation of a multinucleate syncytium (Endo, Phytopathology 53: 622-623, 1963; Mitchum et al., The New Phytologist 199: 879-894, 2013). This highly metabolically active syncytia serve as a nurse cell throughout the rest of the nematode life cycle (Dropkin, Ann Rev Phytopathology 7: 101-122, 1969; Schmitt et al., “Biology and management of soybean cyst nematode.” Marceline, Mo.: Schmitt & Associates of Marceline. xi, 262 p. p. 2004). The process of a syncytia formation is complex and involves nematode directed alterations of plant hormones, metabolic pathways and host gene regulation (Quentin et al., Frontiers in Plant Science 4: 53, 2013). However, since SCN is an obligate pathogen, it must have a functional syncytia to complete its life cycle. The suppression of host plant defense mechanisms is also very important (Abad & Williamson, Advances in Botanical Research, 53: 147-192, 2010). Host plant resistance to nematode infection has several layers, including preformed defenses and specific resistance genes (Williamson & Kumar, Trends Genet 22: 396-403, 2006). SCN possess specific virulence genes that are adapted to evade or suppress multiple types of plant defenses to successfully reproduce (Opperman & Bird, Curr Opin Plant Biol 1: 342-346, 1998). Genetic studies have indicated that resistance to SCN is polygenic in nature and numerous quantitative trait loci (QTLs) have been identified. Even though many SCN resistant cultivars exist, resistance derived from PI88788 and to a lesser extent Peking predominate the commercial seed market (Concibido et al., Crop Science 44: 1121-1131, 2004).

While proteins expressed from esophageal glands are undoubtedly important for nematode parasitism, it has been also suggested that glutathione peroxidases secreted from the hypodermis could protect cyst nematodes against reactive oxygen species (Jones et al., Gene324: 47-54, 2004). Likewise, a lipid binding protein secreted from the cuticle Gp-FAR-1 has been hypothesized to be an inhibitor of jasmonic acid signaling (Prior et al., Biochem J 356: 387-394, 2001). While not secreted proteins, biosynthetic enzymes involved in the production of vitamin B metabolites, pantothenate (VB5), biotin (VB7), thiamin (VB1) and pyridoxal 5-phosphate (VB6) could play a role in circumventing a starvation-based mechanism of nematode resistance (Craig et al., Mol Biol Evol 25: 2085-2098, 2008; Craig et al., J Nematol 41: 281-290, 2009). Two avirulence genes tied to specific host plant resistance genes have been identified in root knot nematodes (Meloidogyne spp.). The map-1 protein is secreted from nematode amphids (Castagnone-Sereno et al., MGG 282: 547-554, 2009; Semblat et al., MPMI 14: 72-79, 2001), while Mj-Cg-1 (Gleason et al., Mol Plant Microbe Interact 21: 576-585, 2008) when silenced via RNAi increased the level of root-knot nematode virulence. Interestingly, Mj-Cg-1 resides in a transposable-element family that may be important for the generation of genetic variation in a asexual nematode species (Gross & Williamson, PLoS ONE 6, 2011).

In cyst nematodes the best examples of avirulence genes are the SPRYSEC effector protein Gp_RBP-1 of Globodera pallida that has been shown to induce a specific hypersensitive reaction when co-expressed with the potato nematode resistance gene Gpa-2 (Sacco et al., PLoS pathogens 5: e1000564, 2009). Likewise, the venom allergen Gr-VAP1 of G. rostochiensis triggers a cell death response in tomato (Lycopersicon esculentum) plants containing the Cf-2 and Rcr3pim genes (Lozano-Torres et al., Proc Natl Acad Sci USA 109: 10119-10124, 2012).

Therefore, it is of particular importance, both to the soybean breeders and to farmers, to have the ability to test soil and identify the residing virulent soybean cyst nematode population. Identification of the biotype will enable a given interested party to employ specific integrated pest management practices for treatment of the soil and/or to develop a planting protocol that includes the identification of resistant soybean cultivars.

SUMMARY

Some phytoparasitic nematodes have the ability to infect and reproduce on plants that are normally considered resistant to nematode infection. Such nematodes are referred to as virulent and the mechanisms they use to evade or suppress host plant defenses are not well understood. Here, we report use of a genetic strategy, allelic imbalance analysis, to associate single nucleotide polymorphisms (SNPs) with nematode virulence on the most common source of resistance used to control Heterodera glycines, the soybean cyst nematode (SCN). To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations growing on resistant and susceptible soybean plants. Three SNPs reproducibly showed SNP allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encodes a biotin synthase (HgBioB) and the other encodes a bacterial-like protein containing a putative SNARE domain (HgSLP-1). The two genes mapped to two different linkage groups. Both genes contained sequence polymorphisms between avirulent and virulent nematodes. In addition, the HgSLP-1 gene was reduced in copy number in virulent nematode populations. The gene encoding HgSLP-1 appeared to produce multiple forms of the protein via intron retention and alternate splicing, but was also part of a gene family.

HgSLP-1 encodes an esophageal-gland protein that is secreted by the nematode during plant parasitism. Silencing expression of the gene by RNAi increased the nematodes' ability to grow on susceptible soybean plants. In bacterial co-expression experiments, HgSLP-1 co-purified with the Rhg1 α-SNAP protein, suggesting this nematode protein binds to this SCN resistance protein. Collectively the data presented herein suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and its absence may be used to evade detection of host defenses.

One of the putative nematode virulence genes aids in the synthesis of vitamin B7, biotin, suggesting part of the nematode resistance mechanism could involve vitamin depravation. The other putative virulence gene encodes a secreted protein that may function in the regulation of membrane fusion; it binds to a soybean protein implicated in causing nematode resistance. The gene that encodes this putative nematode virulence protein was deleted or reduced in copy number in virulent nematode populations, suggesting that the loss of this gene aids the nematode in evading the host-plant resistance gene. The method used to identify these putative nematode genes is expected to be broadly applicable to any plant nematode with a viable genetic system and to the analysis of nematode virulence, or to any measurable nematode phenotype.

In certain embodiments, the present disclosure provides a method for detecting virulent SCN populations. In an example of such methods, the method involves extraction of SCN cysts or eggs from the soil; placing the extracted SCN cysts or eggs in a small container and applying an effective amount of pressure to the extracted cysts or eggs to rupture the cells therein to provide a lysate; to the lysate, a proteinase K (PK) solution can be added to digest the proteins and release the DNA within the lysate, followed by removal of protein fragments and detergents to provide nematode DNA; analyze the DNA, for instance by providing the nematode DNA to a quantitative real-time polymerase chain reaction (QPCR) plate and conducting a QPCR run (e.g., of 1.5 hours) to permit detection of the SCN virulence gene allele frequency. Alternatively, the DNA could be analyzed using direct sequencing, or microarray analysis.

There is provided in a specific example a method, comprising: extracting soybean cyst nematodes (SCN) from a soil sample from a source location (or from an aggregated sample, taken from multiple sub-locations then mixed to obtain a sample more representative of a possibly heterogeneous distribution over the source location area); obtaining DNA from the extracted nematodes; and analyzing the DNA to determine at least one of: (a) the relative copy number of the HgSLP-1 gene in the nematode DNA, and/or (b) the sequence of the HgBioB gene in the nematode DNA, wherein a low relative copy number of HgSLP-1, or the presence of C at position 70 and/or A at position 132 (numbered with reference to SEQ ID NO: 44) within HgBioB, is indicative of SCN virulence in the soil sample. In a further example of such a method, the method further comprises planting in soil at the source location a soybean cultivar having a different source of resistance than that in PI88788 if there is SCN virulence in the soil sample.

Also provided herein is an isolated cDNA molecule, comprising (or consisting of) the nucleic acid sequence shown in SEQ ID NO: 41 (HgSLP-1); or in SEQ ID NO: 50 (HgFAR-1). Additional embodiments are recombinant nucleic acid molecule comprising a promoter sequence operably linked to one of the described nucleic acid molecules, as well as cells transformed with one or more such recombinant nucleic acid molecule(s).

Yet another embodiment is a kit, comprising a container comprising at least one labeled oligonucleotide specific for a HgBioB mutation sequence at position 70 or 132 (numbered with reference to SEQ ID NO: 44).

Another embodiment is a kit, comprising one or more container(s) comprising: a pair of primers specific for HgSLP-1; and at least one labeled oligonucleotide specific for HgSLP-1.

Without intending to be bound by any particular theory, there can be discussion herein of beliefs or understandings of underlying principles or mechanisms relating to embodiments of the disclosure. It is recognized that regardless of the ultimate correctness of any explanation or hypothesis, embodiments of the disclosure can nonetheless be operative and useful.

The foregoing and other objects and features will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. SCN genetic linkage groups containing SCN SNPs linked to virulence. The left column has the map distance in centimorgans and the right column shows the SNP number. The SCN SNPs that show an allelic imbalance when grown on resistant and susceptible soybean plants are indicated with arrows.

FIG. 2. Alignment of paired SOLiD DNA sequencing reads from SCN inbred strain TN20 to the scaffold 385 reference sequence derived from TN10 genomic sequence. The Y-axis shows depth of coverage and the X-axis indicated the base position along the scaffold. The HgSLP-1 gene spans bases 17816 to 21445.

FIG. 3. Quantitative PCR of HgSLP-1 genomic copy number relative to HgFAR-1 in inbred SCN strains, TN10, TN20 OP25, OP20 and OP50.

FIG. 4. Multiple sequence alignment of the HgSLP-1 SNARE domain to related t-SNARE proteins. The ** marks the zero layer residue critical for membrane fusion and * indicates conserved hydrophobic residues in the flanking heptad repeat domains. The following sequences are in the alignment: 1). Locus: 2NPS_B; protein name: chain B; crystal structure of the early endosomal SNARE complex; accession: 2NPS_B; organism: Rattus norvegicus (Norway rat) (positions 1-60 of SEQ ID NO: 30). 2). Locus: HgSLP; protein name: Heterodera glycines SNARE-like protein 1; Accession: organism: Heterodera glycines (soybean cyst nematode) (positions 46-112 of SEQ ID NO: 31). 3). Locus: SYP24_ARATH, protein name: putative syntaxin-24; accession: Q9C615; organism: Arabidopsis thaliana (thale cress) (positions 262-326 of SEQ ID NO: 32). 4. Locus: Q9SML5_CAPAN; protein name: syntaxin t-SNARE; accession: Q9SML5; organism: Capsicum annuum (peppers) (positions 210-275 of SEQ ID NO: 33). 5). Locus: Q8S4W4_PORYE; protein name: Syntaxin PM. Accession: Q8S4W4; Organism: Pyropia yezoensis (marine red alga) (positions 191-256 of SEQ ID NO: 34). 6). Locus: SYP72_ARATH; Protein name: Syntaxin-72; accession: Q94KK6; Organism: Arabidopsis thaliana (thale cress) (positions 170-235 of SEQ ID NO: 35). 7). Locus: BET1L_RAT; protein name: golgi SNARE 15 kDa; accession: 035152; organism: Rattus norvegicus (Norway rat) (positions 12-77 of SEQ ID NO: 36). 8). Locus: SNP30_ARATH; putative SNAP25 homologous protein SNAP30; accession: Q9LMG8; organism: Arabidopsis thaliana (thale cress) (positions 195-260 of SEQ ID NO: 37). 9). Locus: O44419_STRPU; Protein name: Synaptosomal-associated protein 25; accession: O44419; Organism: Strongylocentrotus purpuratus (purple sea urchin) (positions 143-208 of SEQ ID NO: 38). 10). Locus: O01389_HIRME; protein name: SNAP-25 homolog; ACCESSION: 001389; Organism: Hirudo medicinalis (medicinal leech) (positions 144-209 of SEQ ID NO: 39).

FIG. 5. Large read mapping of TN10 cDNA sequences to the HgSLP-1 genomic sequence. The Y-axis shows depth of mapped cDNA coverage (99% identical with 99% overlap of each read) and the X-axis indicates the base position along the HgSLP-1 gene. The numbers mark the exons of HgSLP-1.

FIG. 6A-6D. Immunolocalization of HgSNARE-like protein-1 (HgSLP-1). FIG. 6A-6D are 40× light field images matched with corresponding epifluorescent images of sections of SCN in soybean roots stained using HgSLP-1 antibodies. Arrows point to the basal cell of a subventral esophageal gland in FIG. 6A, the median bulb and esophageal lumen in FIG. 6B and the stylet in FIG. 6C. Panel FIG. 6D shows negative control sections lacking HgSLP-1 antibody staining in the nematode. Arrows in FIG. 6D point to the basal cell of an esophageal gland and the stylet.

FIG. 6D is a composite of two sequential sections from the same nematode. For all light field images, 20 micron scale bars are shown.

FIG. 7. Protein gel blot of HON ARE-like protein and soybean α-NAP protein expressed in E. coli. Proteins in lanes 1-5 were detected using an antibody that binds to HgSNARE-like protein-1 (HgSLP-1). Proteins in lanes 6-9 were detected using an antibody that binds to soybean α-SNAP. Lanes 1 and 6 contain purified protein from E. coli co-expressing full size HgSLP-1 and soybean α-SNAP. Lane 2, 3, 7, and 8 contain independent replicates purified protein from E. coli co-expressing HgSLP-1 missing its signal peptide and soybean α-SNAP. Lanes 4 and 9 contain purified protein from E. coli that only expresses full sized HgSLP-1. Lane 5 contains total protein from E. coli that only expresses full sized HgSLP-1. Protein sizes are shown in kDa.

FIG. 8. HgSLP-1 antibody binding intensity to proteins in gel filtration chromatography fractions containing either HgSLP-1 or both HgSLP-1 and α-SNAP (as indicated).

FIG. 9A-9D. Model for Heterodera glycines SNARE-like protein-1 (HgSLP-1) function. FIG. 9A. During normal plant membrane fusion, soybean v-SNARE proteins (green) interact with t-SNARE proteins (red) and SNAP-25 (yellow) to promote normal membrane fusion, while the NSF (purple) and α-SNAP (blue) are involved in recycling the SNARE proteins. FIG. 9B. In a susceptible interaction HgSLP-1 (black) inhibits membrane fusion by binding to a v-SNARE or soybean α-SNAP. FIG. 9C. In an incompatible interaction the resistant plants over produce the soybean α-SNAP (blue) to bind and neutralize the function of HgSLP-1. FIG. 9D. Virulent SCN lacking HgSLP-1, but containing a variant of the protein (grey), again suppress membrane fusion and promote nematode growth.

FIG. 10 is a graph illustrating HgSLP-1 copy number analysis, from a relative QPCR study of DNA extracted from field isolates of SCN from 14 different locations. The QPCR assay measures the frequency of HgSLP-1 (copy number varies) in each nematode population relative to HgFAR-1 (copy number does not vary). The shorter bars on the graph indicate SCN populations predicted to be the more virulent. The Y-axis shows the ratio of HgSLP-1 and HgFAR-1 (if the two genes are equal in copy number then the ratio is 1). The x-axis shows the sample name (1-14). Samples 1, 7, 9, 10 SCN were able to grow on susceptible and resistant soybean plants (PI88788) and have relatively low copies of HgSLP-1. Series 5 SCN were also able to grow on PI88788 SCN, but had intermediate HgSLP-1 copy number. However, Series 6 SCN, which has a relatively higher copy number of HgSLP-1, did not grow on PI88788, but did grow on a susceptible soybean plant. The other series SCN populations did not grow on either susceptible or resistant soybean, thus their virulence level is unknown.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file named 95855-02_SeqList.txt, created on Feb. 29, 2016, ˜108 KB, which is incorporated by reference herein.

SEQ ID NOs: 1 and 2 are forward and reverse primers used in a SNP assay.

SEQ ID NOs: 3 and 4 are probes used in the same SNP assay.

SEQ ID NOs: 5 and 6 are primers used in SYBR® green Q-PCR.

SEQ ID NOs: 7-14 are RNA sequences used in RNAi silencing of HgSLP-1.

SEQ ID NOs: 15 and 16 are forward and reverse used in detection of HgFAR-1, for instance for copy number assays.

SEQ ID NO: 17 is a probe used in detection of HgFAR-1, for instance for copy number assays.

SEQ ID NOs: 18 and 19 are forward and reverse used in detection of HgSLP-1 (Exon 2), for instance for copy number assays.

SEQ ID NO: 20 is a probe used in detection of HgSLP-1 (Exon 2), for instance for copy number assays.

SEQ ID NO: 21 is the sequence of a peptide used to generate antibodies to HgSLP-1.

SEQ ID NOs: 22 and 23 are forward and reverse primers used to amplify α-SNAP (Glyma18g02590), Accession Number LOC100814639.

SEQ ID NOs: 24 and 25 are forward and reverse primers used to amplify HgSLP-1 cDNA.

SEQ ID NOs: 26 and 27 are forward and reverse primers used to remove the signal peptide from HgSLP-1.

SEQ ID NOs: 28 and 29 are forward and reverse primers used to amplify soybean α-SNAP.

SEQ ID NOs: 30-39 are amino acid sequences of or containing the proteins/fragments aligned in FIG. 4, as follows:

Locus/Accession Protein Name Organism SEQ ID NO: 2NPS_B/ chain B; crystal structure Rattus norvegicus positions 1-60 of 2NPS_B of the early endosomal (Norway rat) SEQ ID NO: 30 SNARE complex HgSLP SNARE-like protein 1 Heterodera glycines positions 46-112 (soybean cyst of SEQ ID NO: 31 nematode) SYP24_ARATH/ putative syntaxin-24 Arabidopsis thaliana positions 262-326 Q9C615 (thale cress) of SEQ ID NO: 32 Q9SML5_CAPAN/ syntaxin t-SNARE Capsicum annuum positions 210-275 Q9SML5 (peppers) of SEQ ID NO: 33 Q8S4W4_PORYE/ Syntaxin PM Pyropia yezoensis positions 191-256 Q8S4W4 (marine red alga) of SEQ ID NO: 34 SYP72_ARATH/ Syntaxin-72 Arabidopsis thaliana positions 170-235 Q94KK6 (thale cress) of SEQ ID NO: 35 BET1L_RAT/ Golgi SNARE 15 kDa Rattus norvegicus positions 12-77 of O35152 (Norway rat) SEQ ID NO: 36 SNP30_ARATH/ putative SNAP25 Arabidopsis thaliana positions 195-260 Q9LMG8 homologous protein (thale cress) of SEQ ID NO: 37 SNAP30 O44419_STRPU/ Synaptosomal-associated Strongylocentrotus positions 143-208 O44419 protein 25 purpuratus of SEQ ID NO: 38 (purple sea urchin) O01389_HIRME/ SNAP-25 homolog Hirudo medicinalis positions 144-209 O01389 (medicinal leech) of SEQ ID NO: 39

SEQ ID NO: 40 (GenBank Accession Number KM575849) is the genomic sequence encoding HgSLP-1, with introns and exons as follows: Exon I, positions 1-141; Exon II, positions 994-1170; Exon III, positions 1412-1471; Exon IV, positions 1616-1756; Exon V, 1856-1954; Exon VI, positions 2514-2642; Exon VII, positions 2832-2927; Exon VIII, positions 3196-3294; and Exon IX, positions 3595-3633 (including the stop codon).

SEQ ID NOs: 41 and 42 are the cDNA sequence of HgSLP-1 and the protein encoded thereby.

SEQ ID NO: 43 is the nucleotide sequence of the Super 385 scaffold; the sequence encoding HgSLP-1 is positions 17,816-21,448.

SEQ ID NO: 44 is the BioB nucleotide sequence, including indications of the SNPs identified herein (position 70: C/G (TN10/TN20); position 132: G/A (TN10/TN20)). SEQ ID NO: 45 is the protein encoded by SEQ ID NO: 44; the SNPs result in variable amino acids at position 24 (Ala or Pro) and position 44 (Arg or Gln).

SEQ ID NOs: 46 and 47 are forward and reverse used in detection of HgSLP-1 (Exon a), for instance for copy number assays.

SEQ ID NO: 48 is a probe used in detection of HgSLP-1 (Exon a), for instance for copy number assays.

SEQ ID NO: 49 is the genomic sequence of HgFAR-1.

SEQ ID NOs: 50 and 51 are the cDNA sequence of HgFAR-1 and the protein encoded thereby.

DETAILED DESCRIPTION I. Abbreviations

BC backcross

BioB biotin synthase

BSA bovine serum albumin

CM chorismate mutase

FAM™ fluorescein fluorescent dye

FD fold difference

Hg Heterodera glycines

HgBioB Heterodera glycines bacterial-like biotin synthase gene

HGT horizontal gene transfer

IPTG isopropyl-beta-D-thiogalactopyranoside

MGBNFQ major groove binder/non-fluorescent quencher

PBS phosphate buffered saline

PK proteinase K

PMSF phenylmethanesulfonyl fluoride

QPCR quantitative polymerase change reaction

Rhg SCN resistance complex genes

RNAi RNA interference

SCN soybean cyst nematode

SHMT serine hydroxymethyltransferase

SLP-1 SNARE-like protein-1

SNAP Soluble NSF Attachment Protein

SNARE SNAP REceptor

SNP single nucleotide polymorphism

TN10 strain of SCN, non-virulent on SCN-resistant soybean plants

TN20 strain of SCN, virulent on SCN-resistant soybean plants

VIC® a fluorescent dye

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:

A coding sequence is the part of a gene or cDNA which codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA.

Complement or complementary sequence means a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules.

Downstream refers to a relative position in DNA or RNA and is the region towards the 3′ end of a strand.

Expression refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of an mRNA into a protein.

An amino acid sequence that is functionally equivalent to a specifically exemplified sequence is an amino acid sequence that has been modified by single or multiple amino acid substitutions, by addition and/or deletion of amino acids, or where one or more amino acids have been chemically modified, but which nevertheless retains one or more characteristics (e.g., biological and/or structural characteristics) of the protein. Functionally equivalent nucleotide sequences are those that encode polypeptides having substantially the same biological activity (or at least one biological activity) as a specifically exemplified protein.

Two nucleic acid sequences are heterologous to one another if the sequences are derived from separate organisms, whether or not such organisms are of different species, as long as the sequences do not naturally occur together in the same arrangement in the same organism.

Homology refers to the extent of identity between two nucleotide or amino acid sequences.

Isolated means altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is isolated, as the term is employed herein.

A nucleic acid construct is a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.

Nucleic acid molecule means a single- or double-stranded linear polynucleotide containing either deoxyribonucleotides or ribonucleotides that are linked by 3′-5′-phosphodiester bonds.

Two DNA sequences are operably linked if the nature of the linkage does not interfere with the ability of the sequences to affect their normal functions relative to each other. For instance, a promoter region would be operably linked to a coding sequence if the promoter were capable of effecting transcription of that coding sequence.

Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided as indicators of virulence or resistance. It is also appropriate to generate probes and primers based on fragments or portions of these nucleic acid molecules. Also appropriate are probes and primers specific for the reverse complement of these sequences, as well as probes and primers to 5′ or 3′ regions.

A probe comprises an isolated nucleic acid attached to a detectable label or other reporter molecule that is not naturally found connected to the nucleic acid. Typical labels include but are not limited to radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. More generally, a label is a composition detectable by (for instance) spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Typical labels include fluorescent proteins or protein tags, fluorophores, radioactive isotopes (including for instance ³²P), ligands, biotin, digoxigenin, chemiluminescent agents, electron-dense reagents (such as metal sols and colloids), and enzymes (e.g., for use in an ELISA), haptens, and proteins or peptides (such as epitope tags) for which antisera or monoclonal antibodies are available. Methods for labeling and guidance in the choice of labels useful for various purposes are discussed, e.g., in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., in Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998). A label often generates a measurable signal, such as radioactivity, fluorescent light or enzyme activity, which can be used to detect and/or quantitate the amount of labeled molecule.

Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other in vitro nucleic-acid amplification methods known in the art.

Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990). Amplification primer pairs (for instance, for use with polymerase chain reaction amplification) can be derived from a known sequence such as the HgSLP-1 or HgFAR-1 or HgBioB sequences described herein, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).

One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 30 consecutive nucleotides of a tyrosine kinase protein encoding nucleotide will anneal to a target sequence, such as another homolog of the designated tyrosine kinase protein, with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise at least 20, 23, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a tyrosine kinase-encoding nucleotide sequences.

Also provided are isolated nucleic acid molecules that comprise specified lengths of tyrosine kinase-encoding nucleotide sequences. Such molecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 or more (e.g., at least 100, 150, 200, 250, 300 and so forth) consecutive nucleotides of these sequences or more. These molecules may be obtained from any region of the disclosed sequences (e.g., a HgSLP-1 or HgFAR-1 or HgBioB nucleic acid may be apportioned into halves or quarters based on sequence length, and isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters, etc.). A cDNA or other encoding sequence also can be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths, and so forth, with similar effect.

Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300 or more consecutive nucleotides of any of these or other portions of a HgSLP-1 or HgFAR-1 or HgBioB nucleic acid molecule, such as those disclosed herein, and associated flanking regions. Thus, representative nucleic acid molecules might comprise at least 10 consecutive nucleotides of the HgSLP-1 (SEQ ID NO: 41), HgFAR-1 (SEQ ID NO: 50) or HgBioB (SEQ ID NO: 44) cDNA.

A polypeptide is a linear polymer of amino acids that are linked by peptide bonds. Promoter means a cis-acting DNA sequence, generally 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription. There can be associated additional transcription regulatory sequences which provide on/off regulation of transcription and/or which enhance (increase) expression of the downstream coding sequence.

A recombinant nucleic acid molecule, for instance a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro through the ligation of two or more non-homologous DNA molecules (for example a recombinant plasmid containing one or more inserts of foreign DNA cloned into at least one cloning site).

Transformation means the directed modification of the genome of a cell by the external application of purified recombinant DNA from another cell of different genotype, leading to its uptake and integration into the subject cell's genome. In bacteria, the recombinant DNA is not typically integrated into the bacterial chromosome, but instead replicates autonomously as a plasmid.

Upstream means on the 5′ side of any site in DNA or RNA.

A vector is a nucleic acid molecule that is able to replicate autonomously in a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g. promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Hence, “comprising A or B” means “including A” or “including B” or “including A and B.” As used herein, “consisting of” excludes any element, step, or ingredient not specified in the aspect element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect.

All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference). References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.

The disclosure may be further understood by the following non-limiting examples. Although the description herein contains many specificities, these should not be construed as limiting the scope of the disclosure but as merely providing illustrations of some of the presently preferred embodiments of the disclosure. For example, thus the scope of the disclosure should be determined by the appended aspects and their equivalents, rather than by the examples given.

While the present disclosure can take many different forms, for the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Any alterations and further modifications of the described embodiments, and any further applications of the principles of the disclosure as described herein are contemplated as would normally occur to one skilled in the art to which the disclosure relates.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended aspects. The specific embodiments provided herein are examples of useful embodiments of the present disclosure and it will be apparent to one skilled in the art that the present disclosure may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

Every formulation or combination of components described or exemplified herein can be used to practice the disclosure, unless otherwise stated.

Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein.

One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this disclosure. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended aspects.

Although the present disclosure has been described with reference to certain embodiments thereof, other embodiments are possible without departing from the present disclosure. The spirit and scope of the appended aspects should not be limited, therefore, to the description of the preferred embodiments contained herein. All embodiments that come within the meaning of the aspects, either literally or by equivalence, are intended to be embraced therein. Furthermore, the advantages described above are not necessarily the only advantages of the disclosure, and it is not necessarily expected that all of the described advantages will be achieved with every embodiment of the disclosure.

III. Overview of Several Embodiments

There is provided in a specific example a method, comprising: extracting soybean cyst nematodes (SCN) from at least one soil sample from a source location (or from an aggregated sample, taken from multiple sub-locations then mixed to obtain a sample more representative of a possibly heterogeneous distribution over the source location area); obtaining DNA from the extracted nematodes; and analyzing the DNA to determine at least one of: (a) the relative copy number of the HgSLP-1 gene in the nematode DNA, and/or (b) the sequence of the HgBioB gene in the nematode DNA, wherein a low relative copy number of HgSLP-1, or the presence of C at position 70 and/or A at position 132 (numbered with reference to SEQ ID NO: 44) within HgBioB, is indicative of SCN virulence in the soil sample. In a further example of such a method, the method further comprises planting in soil at the source location a soybean cultivar having a different source of resistance than that in PI88788 if there is SCN virulence in the soil sample.

In examples of the provided methods, analyzing the DNA to determine the relative copy number of the HgSLP-1 gene in the nematode DNA comprises measuring the abundance of the HgSLP-1 sequence compared to the copy number of a control nematode gene (e.g., HgFAR-1) using quantitative PCR.

Optionally, in embodiments of the provided methods, the method further comprises preparation a ratio of the copy number of HgSLP-1 to the copy number of the control nematode gene, and wherein a ratio of ≤0.2 indicates SCN virulence in the soil. In alternative embodiments, a ratio of ≤0.3, ≤0.35, ≤0.4, ≤0.45, or ≤0.5 indicates SCN virulence in the soil.

In another embodiment of the method, the quantitative PCR uses primer pair SEQ ID NOs: 18 and 19 or NOs: 46 and 47 to amplify HgSLP-1 DNA. By way of example, the copy number of the HgSLP-1 gene in the nematode DNA comprises detecting HgSLP-1 DNA using a labeled probe having the sequence of SEQ ID NO: 20 or 48.

In another embodiment of the method, the quantitative PCR uses primer pair SEQ ID NOs: 15 and 16 to amplify HgFAR-1 DNA. By way of example, the copy number of the HgFAR-1 gene in the nematode DNA comprises detecting the HgFAR-1 DNA using a labeled probe having the sequence of SEQ ID NO: 17.

Also provided herein is an isolated cDNA molecule, comprising the nucleic acid sequence shown in SEQ ID NO: 41 (HgSLP-1); or in SEQ ID NO: 50 (HgFAR-1). In another embodiment, the isolated cDNA molecule has a sequence consisting of the nucleic acid sequence shown in SEQ ID NO: 41 (HgSLP-1); or SEQ ID NO: 50 (HgFAR-1).

Additional embodiments are recombinant nucleic acid molecule comprising a promoter sequence operably linked to one of the described nucleic acid molecules, as well as cells transformed with one or more such recombinant nucleic acid molecule(s).

Yet another embodiment is a kit, comprising a container comprising at least one labeled oligonucleotide specific for a HgBioB mutation sequence at position 70 or 132 (numbered with reference to SEQ ID NO: 44)

Another embodiment is a kit, comprising one or more container(s) comprising: a pair of primers specific for HgSLP-1; and at least one labeled oligonucleotide specific for HgSLP-1. By way of example, in such a kit the pair of primers specific for HgSLP-1 may comprise SEQ ID NOs: 18 and 19 and at least one oligonucleotide specific for HgSLP-1 may comprise SEQ ID NO: 20. Alternatively, the pair of primers specific for HgSLP-1 may comprise SEQ ID NOs: 46 and 47 and at least one oligonucleotide specific for HgSLP-1 may comprise SEQ ID NO: 48. Optionally, such kits for detecting HgSLP-1 may in some embodiments further comprise: one or more additional container(s) comprising: a pair of primers specific for HgFAR-1; and at least one labeled oligonucleotide specific for HgFAR-1, for detecting a control sequence. Alternative, the pair of primers specific for HgFAR-1 may comprise SEQ ID NOs: 15 and 16 and at least one oligonucleotide specific for HgFAR-1 may comprise SEQ ID NO: 17.

IV. Discovery of Soybean Cyst Nematode (SCN) Virulence Genes, and Related Methods

The soybean cyst nematode (SCN), Heterodera glycines, is one of soybean's (Glycine max) most damaging pathogens, causing billions of dollars in annual soybean yield losses (Wrather & Koenning, J Nematol 38: 173-180, 2006). SCN is an obligate parasite that must form a highly metabolically active, multinucleate nurse cell in the plant root (the syncytium) in order to complete its life cycle (Endo, Phytopathology 53: 622-623, 1963; Mitchum et al., The New Phytologist 199: 879-894, 2013; Dropkin, Ann Rev Phytopathology 7: 101-122, 1969′ Schmitt et al., “Biology and management of soybean cyst nematode.” Marceline, Mo.: Schmitt & Associates of Marceline. xi, 262 p. p. 2004). The process of syncytium formation is complex and involves nematode-directed alterations of plant hormones, metabolic pathways and host gene regulation (Quentin et al., Frontiers in Plant Science 4: 53, 2013), in addition to suppression of host-plant defense mechanisms (Abad & Williamson, Advances in Botanical Research, 53: 147-192, 2010). To prevent nematode infection, the plant utilizes several resistance mechanisms, including pre-formed defenses and specific resistance genes (Williamson & Kumar, Trends Genet 22: 396-403, 2006). Understanding the mechanisms host plants use to block nematode parasitism might provide insights into how some nematodes evade these defenses.

Genetic studies have indicated that resistance to SCN is polygenic, and numerous quantitative trait loci (QTLs) for SCN resistance have been identified. However a single accession Plant Introduction (PI) 88788, and to a lesser extent soybean cultivar (cv) Peking, predominate the commercial seed market (Concibido et al., Crop Science 44: 1121-1131, 2004). Recently, resistance genes to SCN were map-based cloned at two loci and both were shown to be atypical plant resistance genes (Cook et al., Science 338: 1206-1209, 2012; Liu et al., Nature 492: 256-260, 2012). The Rhg1 locus from PI88788 resistance was analyzed using an RNA interference (RNAi)-based approach identified three genes that were part of a tandem repeat encoding a soybean α-SNAP protein, a wound inducible protein and a potential amino acid transporter (Cook et al., Science 338: 1206-1209, 2012). The Rhg4 SCN resistance gene from cv Forrest (Peking-type resistance) was recently map-based cloned using targeting induced local lesions in genomes (TILLING), in combination with gene complementation and gene silencing (Liu et al., Nature 492: 256-260, 2012). Rhg4 also encoded a novel type of plant resistance gene, a serine hydroxymethyltransferase (SHMT), which is an enzyme involved in one carbon folate metabolism. The SCN resistance conferred by Rhg4 also requires Rhg1 to function fully, indicating the two seemingly different SCN resistance mechanisms work together in some unknown, but important way (Liu et al., Nature 492: 256-260, 2012).

Plant parasitic nematodes cause considerable damage to agricultural plants throughout the world. The use of naturally occurring, phytoparasitic nematode resistant plants can provide a sustainable, environmentally friendly management strategy. Unfortunately, nematode populations over time can adapt and reproduce on these “resistant plants”. Such nematodes are referred to as virulent or resistance-breaking nematodes. If the molecular mechanism virulent nematodes used to evade or suppress host plant resistance were understood, then virulent populations could be monitored and management strategies could be devised, preserving valuable resistant plant germplasm.

Host-plant resistance is an effective and environmentally friendly management tool. However, virulent nematode populations are able to overcome plant defenses to successfully reproduce on resistant plants (Doyle & Lambert, Mol Plant Microbe Interact 16: 123-131, 2003). These “virulent” SCN are armed with specific virulence genes that have yet to be identified (Dong, & Opperman, Genetics. 146:4 1311-18, 1997; Sereno, Euphytica, 124:2 193-199, 2002; Cook et al., Science 338: 1206-1209, 2012). It is suggested that nematodes probably have to overcome both innate resistance common to many plants, and induced host-plant resistance mechanisms controlled by specific nematode resistance genes (Smant & Jones, “Suppression of Plant Defences by Nematodes.” In: Jones et al., editors. Genomics and Molecular Genetics of Plant-Nematode Interactions. Dordrecht: Springer. pp. 273-286, 2011). In the case of basal resistance mechanisms, plant phytoalexins might be detoxified by esophageal-gland-expressed glutathione-S-transferase (Dubreuil et al., The New Phytologist 176: 426-436, 2007). Likewise, an esophagus-expressed chorismate mutase (CM) is thought to play a similar role by altering the production of chorismate-derived nematode toxins (Doyle & Lambert, Mol Plant Microbe Interact 16: 123-131, 2003; Lambert et al., Mol Plant Microbe Interact 12: 328-336, 1999). Some SCN CM alleles showed a correlation with SCN's ability to reproduce on some SCN-resistant soybean cultivars, suggesting some CM enzymes may aid the nematode in overcoming innate resistance mechanisms (Bekal et al., Mol Plant Microbe Interact 16: 439-446, 2003; Lambert et al., Mol Plant Microbe Interact 18: 593-601, 2005). Other nematode effectors that have been implicated in modulating host defense are GrSPRYSEC-19 (Rehman et al., MPMI 22: 330-340, 2009), Hg30C02 (Hamamouch et al., J Exper Botany 63: 3683-3695, 2012), Hs10A06 (Hewezi et al., Plant Physiol 152: 968-984, 2010), Hs4F01 (Patel et al., J Exp Bot 61: 235-248, 2010) and Mi-CRT (Jaouannet et al., MPMI 26: 97-105, 2013).

Much of how plant parasitic nematodes evade or suppress host plant resistance mechanisms depends on the corresponding plant-resistance genes involved in preventing the nematode from completing its life cycle. Thus, one might expect the unusual nematode resistance genes found at the Rhg1 and Rhg4 loci would require SCN to deploy equally unique mechanisms to overcome these atypical types of resistance. In this document, we describe the use of whole genome allelic imbalance or bulk segregant analysis to identify two candidate SCN virulence genes.

Provided herein are diagnostic assays, which measure copy number of the HgSLP-1 gene in a nematode population. If ratio of a control gene (such as HgFAR-1) to HgSLP-1 in the nematode population is near 1, then that nematode (population) is predicted to not grow on resistant plants where resistance involves a Rhg1 a-SNAP resistance gene. If the ratio is, for instance, 100 times lower (that is, if there is evidence of relative loss of HgSLP-1 gene in the tested nematode population), then the nematode (population) is predicted to grow on resistant soybean. If HgFAR-1 is low in copy number in the population, then it will grow on soybean containing the Rhg1 α-SNAP. If the HsSLP-1 gene has been lost in a (significant) portion of the nematode population, then the nematodes will be able to predate on the plants. If the gene is not present, they will grow on resistant plants containing the Rhg1 α-SNAP. However, soybean varieties having a different source of resistance than this would be predicted to still be resistant.

In another embodiment, to perform a test for the presence or absence of a mutation in a HgBioB sequence of population of nematodes, a suitable genomic DNA-containing sample from a soil site is obtained and the DNA extracted using conventional techniques. The extracted DNA is then subjected to amplification, for example according to standard procedures. The single base-pair mutation(s) in HgBioB is determined by conventional methods including manual and automated fluorescent DNA sequencing, primer extension methods (Nikiforov, et al., Nucl Acids Res. 22:4167-4175, 1994), oligonucleotide ligation assay (OLA) (Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-8927, 1990), allele-specific PCR methods (Rust et al., Nucl. Acids Res. 6:3623-3629, 1993), RNase mismatch cleavage, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), Taq-Man™, oligonucleotide hybridization, and the like.

Sequences surrounding and overlapping single base-pair mutations in the HgBioB gene can be useful for a number of gene mapping, targeting, and detection procedures. For example, genetic probes can be readily prepared for hybridization and detection of the P24_A (position 70 G/C) and R44_Q (position 132 G/A) SNPs. As will be appreciated, probe sequences may be greater than about 12 or more oligonucleotides in length and possess sufficient complementarity to distinguish between the variant sequence and the wildtype, and may optionally be labeled with a detectable, non-naturally occurring label. Similarly, sequences surrounding and overlapping any of the specifically disclosed SNPs, or longer sequences encompassing for instance the entire length of one of the variant HgBioB sequences, or portions thereof, can be utilized in allele specific hybridization procedures.

HgBioB single nucleotide alterations, whether categorized as SNPs or new mutations can be detected by a variety of techniques. The techniques used in evaluating either somatic or germline single nucleotide alterations include allele-specific oligonucleotide hybridization (ASOH) (Stoneking et al., Am. J. Hum. Genet. 48:370-382, 1991), which involves hybridization of probes to the sequence, stringent washing, and signal detection. Other methods include techniques that incorporate more robust scoring of hybridization. Examples of these procedures include the ligation chain reaction (ASOH plus selective ligation and amplification), as disclosed in Wu and Wallace (Genomics 4:560-569, 1989); mini-sequencing (ASOH plus a single base extension) as discussed in Syvanen (Meth. Mol. Biol. 98:291-298, 1998); and the use of DNA chips (miniaturized ASOH with multiple oligonucleotide arrays) as disclosed in Lipshutz et al. (BioTechniques 19:442-447, 1995). Alternatively, ASOH with single- or dual-labeled probes can be merged with PCR, as in the 5′-exonuclease assay (Heid et al., Genome Res. 6:986-994, 1996), or with molecular beacons (as in Tyagi and Kramer, Nat. Biotechnol. 14:303-308, 1996).

Another technique is dynamic allele-specific hybridization (DASH), which involves dynamic heating and coincident monitoring of DNA denaturation, as disclosed by Howell et al. (Nat. Biotech. 17:87-88, 1999). A target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated strand is rinsed away with alkali wash solution. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This probe forms a duplex DNA region that interacts with a double strand-specific intercalating dye. When subsequently excited, the dye emits fluorescence proportional to the amount of double-stranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing temperature of the probe-target duplex. Using this technique, a single-base mismatch between the probe and target results in a significant lowering of melting temperature (T_(m)) that can be readily detected.

A variety of other techniques can be used to detect the mutations in DNA. Merely by way of example, see U.S. Pat. Nos. 4,666,828; 4,801,531; 5,110,920; 5,268,267; 5,387,506; 5,691,153; 5,698,339; 5,736,330; 5,834,200; 5,922,542; and 5,998,137 for such methods. One of ordinary skills will recognize that other techniques for detecting single nucleotide polymorphisms are also applicable.

V. Kits

Kits are provided which contain reagents for determining the (relative) copy number of HgSLP-1, or the presence or absence of mutation(s) in a HgBioB sequence, such as probes or primers specific for the HgSLP-1 gene or a portion thereof, or a HgBioB SNP region, such as those regions surrounding position 70 or position 132. Such kits can be used with the methods described herein to determine whether a sample, such as a soil sample or aggregated soil sample, contains or is contaminated with virulent (or non-virulent) nematodes. The provided kits may also include written instructions. The instructions can provide calibration curves or charts to compare with the determined (e.g., experimentally measured) values.

Oligonucleotide probes and primers, including those disclosed herein, can be supplied in the form of a kit for use in detection of nematodes, or more specifically SCN virulence, in a sample such as a soil sample or aggregated sample. In such a kit, an appropriate amount of one or more of the oligonucleotide primers is provided in one or more containers. The oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers. With such an arrangement, the sample to be tested for HgSLP-1 copy number, or the presence of a HgBioB mutation, can be added to the individual tubes and amplification carried out directly.

The amount of each oligonucleotide primer supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several PCR amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).

A kit may include more than two primers, in order to facilitate the in vitro amplification of HgSLP-1, HgFAR-1, and/or HgBioB sequence(s), for instance.

In some embodiments, kits may also include one or more reagents necessary to carry out nucleotide amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (e.g., polymerase buffer), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).

Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of HgSLP-1, HgFAR-1, and/or HgBioB sequences or mutation(s). In certain embodiments, these probes will be specific for a potential mutation that may be present in the target amplified sequences (e.g., of HgBioB). The appropriate sequences for such a probe will be any sequence that includes one or more of the identified polymorphic sites, particularly nucleotide positions that overlap with the variants shown in position 70 or 132 of SEQ ID NO: 44.

It may also be advantageous to provide in the kit one or more control sequences for use in the amplification reactions. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art; representative controls, based on HgFAR-1, are also described herein.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

EXAMPLES Example 1 A SNARE-Like Protein and Biotin are Implicated in Soybean Cyst Nematode Virulence

Plant parasitic nematodes cause considerable damage to agricultural plants throughout the world. The use of naturally occurring resistant plants can provide a sustainable and environmentally friendly management strategy. However, nematode populations over time can adapt and reproduce on these “resistant plants”. Such nematodes are referred to as virulent. If the molecular mechanism(s) that virulent nematodes use to evade or suppress host plant resistance were understood, virulent field populations could be monitored and management strategies devised, thus preserving valuable resistant plant germplasm.

This study describes the use of a genetic strategy to identify genes in the soybean cyst nematode that allow it to evade or suppress host plant resistance genes. Two potential nematode virulence genes were identified. One gene is involved in the synthesis of vitamin B7 (biotin), suggesting that part of the nematode resistance mechanism overcomes vitamin depravation. The other putative virulence gene encoded a secreted protein that might function in the regulation of membrane fusion. This protein interacted with a soybean protein implicated in plant resistance. The gene that encoded this putative nematode virulence protein was deleted or reduced in copy number in virulent nematode populations, suggesting that the loss of this gene aids the nematode in evading the host-plant resistance. The method used to identify these putative nematode genes is believed to be broadly applicable to any sexually reproducing plant nematode for the analysis of nematode virulence, or to any measurable nematode phenotype. It is believed to be useful for predicting virulence on any plant that uses a-SNAP-type resistance and any nematode that has a gene like HgSLP-1.

Methods

Development of SCN Population for Mapping and Selection

Soybean cyst nematodes, populations TN10 and TN20 were grown by standard methods and cysts were harvested and purified as previously described (Niblack et al., Ann Appl Nematology 25: 880-886, 1993). SCN controlled mating was conducted using a modification of the method described in Dong and Opperman 1997. Briefly, 200 susceptible soybean seedlings (cv Essex unless otherwise stated) were geminated and planted into 50 ml sand filled falcon tubes, that previously had a hole drilled into the bottom and were fitted with an absorbent wick. The tubes containing the seedlings were placed in a tray of water so that the wick would keep the sand uniformly moist throughout the experiment. Soybean seedlings were inoculated with a single inbred SCN strain TN10 J2, which was allowed to parasitize the plant for three weeks. Male SCN were collected by inoculating susceptible plants with J2s from the inbred TN20 SCN strain, and then after a week washing the soil off the roots and placing the plants in a hydroponic culture to collect the males that emerged one week later. Soybean plants were inoculated with SCN TN20 was one week after inoculation of the plants by TN10. To make the controlled cross, the soil was gently rinsed from the seeding inoculated with TN10 and visually inspected to identify SCN females. The plants containing the TN10 females were collected and replanted in sand and then inoculated with TN20 males. After a week, the F₁ eggs were collected and used to re-inoculate a susceptible soybean plant and they were allowed to randomly mate for one generation. Samples of F₁ J2s were also genotyped to verify they all were heterozygous (described below). The F₂ eggs were collected and used to re-inoculate a susceptible plant for one more generation to produce the F₃ SCN eggs, these again were used to infect susceptible plants, but some plants were placed into hydroponic culture to collect F₃ unmated female nematodes for mapping, while others were allowed to mate to produce cysts for F₃ derived single cyst lines used in the allelic imbalance analysis. Eighty four unmated females were harvested and frozen individually in 1.5 ml microcentrifuge tubes and stored at −80° C. until use. The DNA extraction method described in Atibalentja et al. (Mol Genet Genomics 273: 273-281, 2005) was used to extract the DNA from the unmated F3 female SCN.

For genotyping SCN F₁ J2s, individual nematodes were placed in 0.2 ml PCR tubes and a one-step proteinase K DNA extraction method, described in Craig et al. (Mol Biol Evol 25: 2085-2098, 2008), was used to liberate the nematode DNA. The DNA was genotyped using a 2× TAQMAN® master-mix (Life Technologies) following manufacturer recommendations. The SNP assay, run on an Applied Biosystems (Foster City, Calif.) 7900HT Sequence Detection System under recommended settings using the following primers and probes: F-primer: GCGGCAGATTGAAGAAGCATTT (SEQ ID NO: 1), R-primer: GCACGGCACTGATCAGACA (SEQ ID NO: 2), Probe: FAM-CCTCTCCATGCGGACC-MGBNFQ (SEQ ID NO: 3; SNP underlined), VIC-AGCCTCTCCATACGGACC-MGBNFQ (SEQ ID NO: 4; SNP underlined). (“FAM™” and “VIC®” are reporter dyes; “MGBNFQ” is the major groove binder/non-fluorescent quencher.) Standard PCR conditions were used for the TAQMAN® assays: 50° C. for 10 min, followed 95° C. for 10 minutes, then 40 cycles of 95° C. for 10 sec, and 60° C. for 1 min.

Selection of SCN Populations on Resistant and Susceptible Plants

Single cyst SCN lines were allowed to grow for two generations on susceptible soybean (Essex) and then ten lines were harvested and equal amounts of eggs pooled. Two pools of ten SCN F₃-derived lines were produced. Five SCN resistant (Rhg1) backcross 3 (BC3) and five susceptible BC3 soybean plants were inoculated with equal numbers of the pooled eggs and the nematodes were allowed to reproduce for one generation. One month later, the second pool of SCN eggs was used to inoculate a second set of five SCN resistant (Rhg1) backcross 3 (BC3) and five susceptible BC3 soybean plants. This set of plants served as a biological replicate for the allelic imbalance experiment.

For both experiments, the resulting cysts were harvested as described above, and approximately 50-100 cysts from each plant were placed into 1.5 ml microcentrifuge tubes, frozen in liquid nitrogen, pulverized with a steel pestle and then the DNA was extracted using a DNeasy tissue kit (Qiagen, Valencia Calif.) following the manufacturer's instructions. The extracted genomic DNA (50 μl) was precipitated by adding 20 μg of yeast tRNA, 1 μl of pellet paint NF, 5 μl of 3 M Na acetate to the genomic DNA, followed by 2.5 volumes of cold 100% ethanol. The resulting DNA precipitate was collected by centrifugation and washed with 70% ethanol, and then air-dried. The DNA was amplified using a GENOMIPHI™ DNA amplification kit following the manufacturer's recommendations (GE Healthcare, Piscataway N.J.). The resulting DNA was treated with EXOSAP-IT® PCR cleanup reagent to remove primers and nucleotides following manufacturer's instructions (Affymetrix). For all DNA samples the SCN DNA concentration was determined by SYBR® green QPCR using 2×SYBR® green master-mix (Life Technologies, Grand Island N.Y.), and primers GCCATTGGAGCGCCAGATGC (SEQ ID NO: 5) and GGCTCATCGGCGGCACAA (SEQ ID NO: 6). Standard PCR conditions were used for the TAQMAN® assays: 50° C. for 10 min, followed 95° C. for 10 minutes, then 40 cycles of 95° C. for 10 sec, and 60° C. for 1 min, followed by a melting curve cycle. A standard curve of SCN DNA with known concentration was used to calculate the absolute concentration of the amplified SCN DNA.

Allelic Imbalance Analysis

The SNP sequences were identified by comparing TN10 SCN cDNA sequences to SCN TN20 genomic DNA sequence. The TN10 cDNAs derived from J2 RNA (1,949,251 sequence reads) and egg RNA (3,080,637 sequence reads) were produced by a 454 GS FLX sequencer at the University of Illinois, Roy J Carver Biotechnology Center, and were assembled into contigs using the de novo assembly program in the CLC Genomics Workbench (CLCbio, Boston, Mass.). The TN10 contig sequences were concatenated into one million base lengths and used as reference sequences for the alignment of 577,902,089 TN20 genomic sequences (paired-end 25 nucleotide reads) generated on the SOLiD™ sequencing platform (SeqWright Inc., Houston Tex.). SNPs were identified in the alignments and selected if the aligned TN20 reads were different from the TN10 cDNA reference sequence, if the coverage was between 20 and 50 reads deep and if there were not other SNPs within 100 bp. Selected SNPs were also confirmed by alignment with TN10 genomic sequences, also produced on a SOLiD™ sequencing platform as described above. A list of 1,536 SNPs was sent to Illumina (San Diego, Calif.) to synthesize the GoldenGate genotyping oligonucleotides. The SCN DNA was diluted to a concentration of 100 ng/μl and 50 μl was sent for genotyping. The GoldenGate genotyping was conducted at the University of Illinois Functional Genomics Laboratory in the Roy J. Carver Biotechnology Center following standard protocols. After genotyping the SCN genotypes and the theta values for each SNP and DNA sample were assigned using GENOMESTUDIO® V2011.1 (Illumina Inc.). The SCN genetic linkage groups were produced by downloading the SNP data from the GENOMESTUDIO® data analysis software program into Microsoft Excel where the SNPs were placed into phase with the parental nematode genotypes. The SCN genotypes, from 723 SNPs, were imported into MST Map (Wu et al., “Efficient and Accurate Construction of Genetic Linkage Maps from Noisy and Missing Genotyping Data.” Philadelphia Pa. 395-406 p. 2007) to produce linkage groups. The following settings were used: population type RIL3, population_name LG, distance_function kosambi, cut_off_p_value 1.0×10⁻¹³, no_map_dist 15.0, no_map_size 3, missing_threshold 0.25, estimation_before_clustering no, detect_bad_data yes, objective_function ML, number_of_loci 723, number_of_individual 84. The maps were drawn using the map draw macro in Microsoft Excel (Liu & Meng, Yi chuan 25: 317-321, 2003).

Annotation of SCN Scaffolds Containing Selected SNPs Linked to Virulence

The DNA sequence for each SNP that showed allelic imbalance was used to identify a SCN genomic sequence scaffold using BLASTN. The SCN scaffolds, build 1, were obtained from the JGI Heterodera glycines community genome sequencing project (available online at genome.jgi.doe.gov/). The JGI SCN genome is from SCN inbred strain TN10. The sequence data was used to make and search a BLASTN database within the CLC Genomics Workbench. SCN scaffolds that matched the SNPs were annotated by performing a large-gap read mapping using SCN cDNA sequence derived from 454 and Illumina sequencing platforms. The mapped cDNAs were used to define the beginning and end of the expressed genes on the transcripts and intron sequences were removed to produce a final cDNA sequence. All expressed genes on the scaffold were compared to protein sequences in the databases via BLASTX. The cDNA sequence for HgSLP-1 was confirmed by PCR amplifying the full-length cDNA using PCR primers flanking the open reading frame. To do this, TN10 RNA was extracted and converted to cDNA as described in Craig et al. (Mol Biol Evol 25: 2085-2098, 2008). The cDNA was amplified a proof reading thermostable DNA polymerase and then cloned into pCR2.1 plasmid vector. The DNA sequenced was determined at the University of Illinois Roy J. Carver Biotechnology Center. To identify DNA polymorphisms in the candidate virulence genes, genomic DNA sequence derived from SCN strain TN20 was mapped to each scaffold using the CLC Genomics Workbench and SNPs were identified using the quality-based variant detection program. The coiled-coil domain was identified using the program COILS on the ExPASy web site (Lupas et al., Science 252: 1162-1164, 1991). The t-SNARE domain was detected and the multiple sequence alignment was produced using NCBI's conserved domain database (Marchler-Bauer et al., Nucleic Acids Res 41: D348-352, 2013), but the conserved, zero-layer polar amino acid residue with the flanking hydrophobic amino acid heptad repeats were detected using hydrophobic cluster analysis (Callebaut et al., CMLS 53: 621-645, 1997). The signal peptide was predicted using the TargetP 1.1 server (Emanuelsson et al., Nature Protocols 2: 953-971, 2007), also found on the ExPASy web site.

Silencing of HgSLP-1 by RNAi

SCN inbred strain TN10 was used for RNAi experiments. Four DsiRNAs directed against HgSLP-1 were synthesized by Integrated DNA Technologies (Coralville, Iowa) and were used at a concentration of 200 pmoles per ml. SCN J2s, 10,000/ml were treated with the DsiRNAs in 50 mM octopamine in M9 media (Urwin et al., Mol Plant Microbe Interact 15: 747-752, 2002) overnight with rotation and were then inoculated on to plants and allowed to reproduce for 30 days, then the number of cysts that formed were counted. The experiment was repeated with similar results. NC1 negative control DsiRNA sequence (DS NC1) was obtained from Integrated DNA Technologies and used at the same concentration and conditions described above. Four sections of the gene transcript were targeted.

1S:  (SEQ ID NO: 7) 5′-UCUAAUAGUGAUAACAGCCUGCUACUU-3′ 1AS: (SEQ ID NO: 8) 3′-AGAUUAUCACUAUUGUCGGACGAUG-5′ 2S:  (SEQ ID NO: 9) 5′-AGACAAUAUGAGAACAUCGUGCCACAU-3′ 2AS:  (SEQ ID NO: 10) 3′-TCUGUUAUACUCUUGUAGCACGGUG-5′ 3S:  (SEQ ID NO: 11) 5′-GUUCAUUUCAUCUCGAAGCUCGUCGAU-3′ 3AS:  (SEQ ID NO: 12) 3′-CAAGUAAAGUAGAGCUUCGAGCAGC-5′ 4S:  (SEQ ID NO: 13) 5′-UGGUCGAGUCGUUGGUCUGUUCGGUCC-3′ 4AS:  (SEQ ID NO: 14) 3′-ACCAGCUCAGCAACCAGACAAGCCA-5′

In the susceptible line (TN10), when treated with RNAi to inhibit expression of HgSLP-1, they grow better. However, knocking down in expression did not have an effect on a resistant plant (TN20). This experiment did not show an actual RNA difference.

Copy Number Analysis for HgSLP-1

SCN genomic DNA was extracted as described above for SCN strain TN10, TN20, OP20, OP25 and OP50. Two TAQMAN® probe-based gene expression assays were used to measure the copy number of the HgSLP-1 gene relative to a control gene, HgFAR-1 (SEQ ID NO: 49). Standard PCR conditions were used for the TAQMAN® assays: 50° C. for 10 min, followed 95° C. for 10 minutes, then 40 cycles of 95° C. for 10 sec, and 60° C. for 1 min. For each DNA type, triplicate PCR reactions were conducted and the entire experiment was conducted twice with similar results. The data were compared using the ΔCt method (Livak & Schmittgen, Methods 25: 402-408, 2001), where ΔCt=Ct^(target)−Ct^(control gene) and the fold difference (FD) between the HgSLP-1 target gene and the HgFAR-1 control gene was calculated using the equation, FD=2^(−ΔCt). The fold differences were normalized to OP25 since there was nearly no difference in ΔCt for this SCN population. HgFAR-1 (SEQ ID NO: 49) was known to be of consistent copy number Craig et al. (Mol Biol Evol 25: 2085-2098, 2008). The primer and probes for detection of HgFAR-1 were as follows: F-primer AGGTGACCAAATTCTACC (SEQ ID NO: 15), R-primer GGGTGTCCATTTATTTGC (SEQ ID NO: 16), Probe FAM-CTGACCGAGGATGGACAA-MGBNFQ (SEQ ID NO: 17). The HgSLP-1 TAQMAN® primers and probes detected sequences in the first intron of HgSLP-1, the following oligonucleotides were used: F-primer CGAGATGAAATGAACCAAA (SEQ ID NO: 18), R-primer GAGTCGTTTGTCCATTTG (SEQ ID NO: 19), Probe FAM-AACACGAGATTGGAC-MGBNFQ (SEQ ID NO: 20). Alternatively, the following HgSLP-1 TAQMAN® primers and probes are useful for detecting sequences in the first intron of HgSLP-1: F-primer TAGGCCCTGCCATATTTG (SEQ ID NO: 46), R-primer TCCTGTGTGTTTCAAGTG (SEQ ID NO: 47), Probe FAM-CAAGGAATGGGCTCAA-MGBNFQ (SEQ ID NO: 48).

Localization of HgSLP-1 Protein in SCN-Infected Soybean Roots

Antibodies to HgSLP-1 were generated from a synthetic peptide (CRHLFESGEASETAS; SEQ ID NO: 21) in rabbits and affinity purified by GenScript (Piscataway, N.J.). Susceptible soybean seedlings (Essex) were inoculated with inbred SCN strain TN10 eggs, which were allowed to hatch and infect the soybean roots for 5 days to generate a mixed age population of nematodes. Root sections containing SCN infection sites were dissected and 0.5 cm long root segments were placed in ice-cold FAA fixative (50% ethanol, 5% acetic acid, 10% formalin). The nematodes and roots were microwave-fixed and embedded in paraffin (Lambert et al., Mol Plant Microbe Interact 12: 328-336, 1999). Immunolocalization of HgSLP-1 was conducted by sectioning (10 μm) the paraffin embedded roots and mounting them on Probe-on-Plus slides (Fisher Scientific, Pittsburgh, Pa.) over night at 42° C. The slides were soaked twice in xylene for 5 minutes, and then incubated in 100% acetone for 5 minutes. After repeating the acetone incubation the slides were hydrated by soaking for 5 minutes each in 95%, then 85%, 70%, and 50% acetone. Finally they were soaked in distilled H₂O and phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, KH₂PO₄ pH 7.4) for 5 minutes each. The nematodes were permeabilized by incubating the nematodes in PBS containing 4 μg/ml proteinase K for 30 minutes. The proteinase K was inactivated by soaking the slides 5 minutes in PBS with 0.2% glycine, then in PBS with 1 mM phenylmethanesulfonyl fluoride (PMSE), followed by a 5 minute treatment in PBS. The slides were blocked with 10% goat serum in PBS for 30 minutes, and then washed with PBS with 0.1% bovine serum albumin (BSA) for 5 minutes. The slides were incubated for two hours with the HgSLP-1 add primary antibody or pre-immune serum as a negative control (1:250 dilution) in PBS with 0.1% BSA, then washed 3 times, 10 minutes each wash, in PBS with 0.1% BSA. Next, the slides were incubated with the secondary antibody, goat-anti-rabbit labeled with OREGON GREEN® 488 dye (Life Technologies) in PBS with 0.1% BSA (1:250 dilution) and incubated for 2 hours. Finally, the slides were washed 3 times (10 minutes each wash) in PBS with 0.1% BSA. The slides were washed once with water and mounted with a coverslip using Prolong anti-fade and allowed to dry overnight. The sections were observed on a Zeiss Axio Scope 2 florescence microscope and digital images were captured with a Zeiss Axiocam. These experiments were conducted three times with similar results.

Production of α-SNAP Antibodies

Total RNA from soybean cv Forrest was converted to cDNA and used as a template to PCR amplify α-SNAP (Glyma18g02590) accession number LOC100814639 using primers XmaI-SNAP-F: AAACCCGGGAATGGCCGATCAGTTATCGAAGG (SEQ ID NO: 22) and XhoI-SNAP-R: AAAACTCGAGTCAAGTAATAACCTCATACTCC (SEQ ID NO: 23). The resulting PCR product was cloned between XmaI and XhoI cloning sites in the pGEX-5x-1 vector that adds a GST-tag (pGEX-5x-1-SNAP-GST). The construct was confirmed to be correct by sequencing (Genewiz, South Plainfield, N.J.). The construct plasmid was transformed into E. coli BL21 and sent to Rockland Immunochemicals (Gilbertsville, Pa.) where the company produced and purified the protein and then injected it into rabbits. The SNAP polyclonal antibody was affinity purified and cross adsorbed to remove antibodies that might bind to the GST tag.

Characterization of HgSLP-1 by Co-Expression/Co-Purification

The HgSLP-1 protein sequence (SEQ ID NO: 42) derived from SCN strain TN10 and the susceptible allele of the soybean α-SNAP were used to synthesize the coding region using DNA STRINGS™ DNA (Life Technologies). HgSLP-1 cDNA (SEQ ID NO: 41) was PCR amplified using primer HgSLP1F-BglII: GGTCTTGAGCGGATATCTTAACCGG (SEQ ID NO: 24) and primer HgSLP1R-EcoRV: CTCGACATCTCAGATCTATGGCACC (SEQ ID NO: 25) which had had terminal start-BglII and EcoRV-end sites for cloning. The cDNA was PCR amplified using CloneAmp HiFi PCR premix (Clontech, Mountain View Calif.) and 10 pmoles of each primer using 98° C., 10 sec and 25 cycles of 98° C. for 10 sec, 60° C. for 15 sec, 72° C. for 10 sec followed by a 72° C. for 2 min. The PCR products were gel purified and the cDNA insert was digested with the restriction enzymes BglII and EcoRV and ligated into the second expression position of the vector pCDF Duet-1 (Novagen, San Diego, Calif.) digested with the same enzymes. The pCDF Duet-1-HgSNP-1 vector was then transformed into E. coli Top10 cells. Selection was performed on spectinomycin (50 mg/ml) LB agar plates. The resulting plasmid containing HgSLP-1 was verified for accuracy by sequencing the nematode gene at the University of Illinois Roy J. Carver Biotechnology Center. Later the HgSLP-1 plasmid was transformed into JM109DE3 E. coli and used as a negative control that expresses only the HgSLP-1 protein.

The signal peptide was removed from the HgSLP-1 gene in the pCDF-Duet-1 vector by PCR amplifying the plasmid with primers HgSLP-1deltaspF: GAAAAAGCAGCACCGAATGC (SEQ ID NO: 26) and HgSLP-1deltaspR: CGGTGCTGCTTTTTCCATAGATCTGCCATATGTATATCTCCT (SEQ ID NO: 27). The PCR was conducted using CLONEAMP™ HiFi PCR premix as described above, except that the extension time was 30 seconds. The resulting PCR product was treated with cloning enhancer, the plasmid was circularized using the In-Fusion HD enzyme premix and was transformed into E. coli Stellar competent cells following the manufacturers protocol (Clontech). The resulting plasmid containing HgSLP-1 missing the signal peptide was verified for accuracy by sequencing the nematode gene as described above.

The cDNA of the soybean α-SNAP was synthesized using DNA Strings (Life Technologies). The cDNA was PCR amplified using primers soysnapF-EcoRI: TCGCGAATGCGAATTCATGGCCGAT (SEQ ID NO: 28) and soysnapR-SalI: CGTCGAGCATGTCGACTCAATGGTG (SEQ ID NO: 29) contained start-EcoRI and end-SalI sites. The cDNA was digested with EcoRI and SalI and ligated into the first expression position of pCDF Duet-1-HgSLP-1. The plasmid was then transformed into JM109DE3 E. coli that contains the T7 RNA polymerase gene and allows isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the lac-T7 promoters on pCDF-Duet-1. For all protein expression experiments, the E. coli containing the construct was grown at 37° C. until OD_(600 nm) was 0.4, then it was induced with 1 mM IPTG for 4 hours at 30° C. The induced E. coli cells were collected by centrifugation, suspended in 50 mM sodium phosphate (pH 7.4), 150 mM NaCl containing 1× Halt Protease Inhibitor cocktail, lacking EDTA (Thermo Scientific) and lysed using a B-PER bacterial protein extraction kit (Fisher Scientific) following the manufacturer's instructions. The protein concentration of the resulting E. coli protein extract was determined using the Pierce BCA protein assay kit (Fisher Scientific). Equal concentrations of proteins from the negative control and experimental samples were purified using Dynabeads His-Tag Isolation kit (Life Technologies) following manufacture's protocol. The eluted proteins were run on a Mini-Protean TGX SDS-PAGE 4-20% gradient gel (BioRad, Hercules Calif.) and the proteins were transferred to a nitrocellulose membrane using an iBlot dry blotting system (Life Technologies). The protein blot was incubated with blocking buffer (0.1 M maleic acid, 0.15 M NaCl, 1% BSA, 0.3% Triton X-100) for one hour. The primary polyclonal antibodies, anti-HgSLP-1 or anti-α-SNAP, were diluted 1:5000 in blocking buffer and incubated with the blot for 30 minutes and were then washed with blocking buffer three time for 10 minutes each. The secondary antibody, goat anti-rabbit alkaline phosphatase conjugate, was also used at a 1:5000 dilution in blocking buffer and was incubated and washed as described above. The secondary antibody was detected using Western Blue Stabilized substrate for alkaline phosphatase (Promega, Madison Wis.) and the reaction was stopped using TE (10 mM Tris-HCL, 1 mM EDTA) after approximately 20 minutes of development at room temperature. The experiment was repeated with similar results.

The total E. coli protein extracts expressing HgSLP-1 alone or both HgSLP-1 and α-SNAP were analyzed by gel filtration chromatography. The gel filtration column was 50 cm tall and had a diameter of 1.8 cm. The column was packed with SUPERDEX™ 75 prep grade resin (Amersham Biosciences) and a Bio-Rad ECONO™ gradient pump and model 2110 fraction collector were used to pack and collect the protein fractions. The column was equilibrated using 50 mM sodium phosphate (pH 7.4), 150 mM NaCl buffer and then calibrated using a LMW gel filtration calibration kit (Amersham Biosciences). Blue dextran 2000 was used to determine the void volume of the column and ribonuclease A (13.7 kDa), chymotrypsinogen A (25.0 kDa), ovalbumin (43.0 kDa), albumin (67.0 kDa) were used in the column calibration. For each column run, the flow rate of the column of 0.25 ml/minute and each fraction was collected for two minutes. Before each sample was added, 200 μl of blue dextran 2000 was added to the column, allowed to run into the column for one minute, then 200 μl of protein extract was added (either HgSLP-1 or the extract containing both HgSLP-1 and α-SNAP). The extracts were the same ones used in the co-purification experiments described above. The fractions obtained were tested for the presence of HgSLP-1 using a protein dot blots. Briefly, 400 μl of each fraction was precipitated by adding 1600 μl of cold acetone. The proteins were allowed to precipitate on ice for 30 minutes, then they were centrifuged and the acetone was removed from the protein pellet. The proteins were dried in a SPEEDVAC™ concentrator and then suspended in 10 μl of 1×SDS PAGE buffer and boiled for 5 minutes. 2 μl of the protein fraction was spotted onto nitrocellulose, dried, and then the HgSLP-1 was detected as described above. The resulting spots were quantified using NIH image and the resulting intensities were plotted using Microsoft excel. The experiment was repeated with similar results.

DNA and RNA Sequence Data

The HgSLP-1 gene (genomic) sequence is deposited in GenBank Accession Number KM575849 (40). All next-generation DNA sequence data used in this project is deposited in the NCBI BioProject 680464 titled, “Heterodera glycines genome sequencing”.

Genomic Sequence:

SCN strain TN20 SOLiD™ sequencing platform (2×25 base 3 kb mate pair) 577,902,089 reads.

SCN strain TN10 SOLiD™ sequencing platform (50 base) 270,363,891 reads.

RNAseq:

SCN strain TN10 454 sequencing platform: J2 RNA (1,949,251 sequence reads) and egg RNA (3,080,637 sequence reads) 200-500 base.

SCN strain TN10 Illumina sequencing platform (2×75 base paired end) 263,530,527 reads. SCN strain TN10 Illumina sequencing platform (2×100 base paired end) 365,288,386 reads.

SCN SNPs:

1536 SNP and flanking sequence.

DNA sequences for scaffolds: 385 (SEQ ID NO: 43), 1924 and 20.

Results

Allelic Imbalance Analysis:

Dong & Opperman (Genetics 146: 1311-1318, 1997) established that genetic analysis of SCN virulence was feasible. The subsequent development of high throughput DNA sequencing and genotyping methods made a map-based approach to identify SCN virulence genes possible. In this project we constructed an F₃ mapping population of SCN, segregating for virulence, and then used an allelic imbalance/bulk segregation-based approach to identify regions of the SCN genome containing virulence gene candidates.

To create the SCN mapping population, two inbred SCN strains were crossed. The female parental strain, TN10, was non-virulent and the male parental strain, TN20, was virulent on SCN resistant soybean. The resulting F₁ nematodes were allowed to randomly inter mate for two generations to generate a mapping population of unmated F₃ female SCN and F₃ single cyst derived populations to use for allelic imbalance analysis.

A pool of F3 single cyst derived populations was used to inoculate soybean plants containing the Rhg1 resistance locus, or susceptible plants harboring only the susceptible Rhg1 alleles. The resulting cysts were harvested from all plants and the DNA was extracted. The SCN populations used in the selection experiments contained both virulent and avirulent SCN, thus one would expect the frequency of SCN virulence genes to increase in SCN populations grown on soybean harboring the Rhg1 resistance locus, but not on the susceptible plants. Furthermore, genetic recombination in the SCN genome should create a condition in the SCN population where allelic ratios of single-nucleotide polymorphisms (SNPs) in or near SCN virulence genes should be altered, but SNPs physically farther away from or unlinked to virulence genes will not have distorted ratios of SNP alleles in comparison to the susceptible control. This type of allelic imbalance or bulk segregant analysis can be a useful genetic approach if a way of conducting high-throughput SNP analysis is available.

Since commercial genotyping tools were absent for SCN, a custom Illumina SNP array was developed. At the time of the initiation of this project, the SCN genome was not available, so cDNA sequence reads of SCN inbred strain TN10 from egg and J2 developmental stages were collected, assembled and used as a template to identify SNPs in the SCN genome. SNPs that differed between the parental strains were identified by aligning genomic sequence collected from both parental inbred strains. A total of 1536 SNPs were selected that were homozygous between the parental SCN inbred strains and these were used to generate custom genotyping oligonucleotides for the Illumina GoldenGate genotyping system.

Two types of SCN DNA were genotyped, one was a set of 10 DNA samples for the allelic imbalance analysis and the other was a set of 84 DNA samples extracted from the F₃ mapping population. Parental DNA from SCN strain TN10 and TN20 were run as controls. The experiment was repeated to provide a biological replicate. In the allelic imbalance analysis, out of the 1536 SNPs tested, three SNPs showed a statically significant imbalance where the virulent and avirulent SNP allele consistently differed in frequency when the bulk SCN populations were grown on susceptible and resistant plants. Thus, the three SNPs (212, 1063 and 1533) were considered good candidates for markers linked to SCN virulence loci (Table 1). A partial genetic map containing these three SNP markers was constructed from the SCN F₃ mapping population SNP data and showed that the three virulence associated SNPs mapped to two different linkage groups (FIG. 1).

TABLE 1 Theta values for allelic imbalance analysis SNP 212 SNP 1533 SNP 1063 Suscep- Resis- Suscep- Resis- Suscep- Resis- tible tant tible tant tible tant Experi- ment 1^(a) Rep 1^(b) 0.1894^(a) 0.3439 0.5882 0.6592 0.3201 0.3737 Rep 2 0.2335 0.4187 0.607 0.6850 0.2509 0.3860 Rep 3 0.2634 0.4705 0.5903 0.6775 0.2924 0.3765 Rep 4 0.2702 0.4609 0.4251 0.7285 0.2070 0.3830 Rep 5 0.3180 0.4698 0.4775 0.6734 0.3694 0.3508 Mean 0.2549 0.4327 0.5376 0.6847 0.2879 0.374 Std Dev^(d) 0.0475 0.0540 0.0813 0.0262 0.0624 0.0138 P^(e) 0.00014 0.00214 0.00243 Experi- ment 2 Rep 1 0.2262 0.3309 0.4815 0.5297 0.1614 0.2084 Rep 2 0.2201 0.3086 0.4933 0.5238 0.1779 0.1796 Rep 3 0.1691 0.3076 0.4288 0.5422 0.1450 0.2192 Rep 4 0.1727 0.3484 0.3894 0.5090 0.1474 0.1812 Rep 5 0.1713 0.2608 0.4208 0.5984 0.1399 0.2389 Mean 0.1919 0.3112 0.4427 0.5406 0.1543 0.2054 Std Dev 0.0286 0.0329 0.0435 0.0344 0.0154 0.0253 P^(c) 0.00014 0.00214 0.00243 ^(a)Experiment 1 and 2 are replicates of the entire experiment; ^(b)Rep 1-5 are technical replicates within each experiment; ^(c)Significant differences in theta values for SNPs from SCN populations grown on susceptible and resistant soybean lines indicate allelic imbalances; ^(d)Std Dev = standard deviation; ^(e)P = probability from one-tailed Student's T-Test.

Identification of Linked Candidate Virulence Genes Via Homology and Polymorphisms:

To examine the genomic regions containing the SNPs associated with virulence, a SCN genome sequence was required. Fortunately, a draft assembly of the SCN TN10 genome was recently completed by the Joint Genome Institute (JGI). BLASTN was used to match the DNA sequence flanking the SNPs to the genome scaffolds. The BLASTN search identified three scaffolds; scaffold 385 (40,259 bp; SEQ ID NO: 43) for SNP 212, scaffolds 1924 (15,316 bp) for SNP 1063 and scaffold 20 (176,619 bp) for SNP 1533.

Although it was unknown how close the SNPs might be to candidate SCN virulence gene(s), it was thought that a closely linked SCN virulence gene could be identified based upon homology to known pathogenicity related proteins and the presence of sequence polymorphisms between virulent and avirulent SCN.

Since the SCN scaffolds were from a preliminary build of the SCN genome, they were not annotated. To identify expressed genes, SCN transcriptome DNA sequence, derived from egg and J2 RNA, was aligned to the scaffolds. The beginning and end of the expressed genes were identified and intron sequences were removed. The resulting cDNA sequences were then compare to known proteins using BLASTX (Table 2). Plant parasitic nematodes acquire genes via horizontal gene transfer (HGT) from microorganisms, which often plan a role in plant pathogenesis (Craig et al., Mol Biol Evol 25: 2085-2098, 2008; Smant et al., Proc Natl Acad Sci USA 95: 4906-4911, 1998), thus any HGT candidates on the scaffolds were given extra scrutiny. On two of the scaffolds, potential HGT candidates were identified. Scaffold 385 (SEQ ID NO: 43) contained a gene with homology to a bacterial protein from Paenibacillus dendritiformis, as well as a 162 amino acid fragment of a unpublished putative dorsal esophageal gland protein, Ha-dsl-1, from Heterodera avenae (AD182806.1).

TABLE 2 Protein homology of expressed genes on SCN genome scaffold 385 Size e- Seq. (bp) Protein match Organism Accession # Value A 349 No significant similarity B 2822 No significant similarity C 787 No significant similarity D 1756 Phosphoglycerate mutase Haemonchus CDJ82788.1 1e−16 domain containing protein contortus E 1000 Zinc finger BED domain- Ciona intestinalis XP_004227062.1 3e−08 containing protein F 978 Protein of unknown Paenibacillus WP_006677173 8e−06 function dendritiformis G 2871 Dorsal esophageal gland Heterodera avenae ADI82806 6e−24 protein H 539 Protein of unknown Haemonchus CDJ97289 1.9e−02   function contortus I 2109 Protein of unknown Ascaris ERG83921.1 5e−04 function J 1262 No similarity K 2348 Protein of unknown Necator ETN70279.1 3e−09 function americanus L 2748 Regulator of nonsense Haemonchus CDJ83790.1 3e−12 transcript contortus

The Ha-dsl-1 protein is 463 amino acids in length, however, only 62 amino acids of the N-terminus of the SCN Ha-dsl-1-like protein was identified in scaffold 385. The remaining 100 amino acids of the predicted SCN Ha-dsl-1-like protein were not similar to Ha-dsl-1. Furthermore, when genomic sequences from SCN TN10 and TN20, collected from an Illumina sequencer, were aligned to the SCN Ha-dsl-1-like gene, the same allelic form was present in both SCN TN10 and TN20, thus this gene was not considered a promising virulence gene candidate.

However, the SCN Paenibacillus dendritiformis-like protein in scaffold 385, which spans from base positions ˜18,000 to 22,000 in the scaffold (SEQ ID NO: 43), appeared more interesting. When Illumina TN10 and TN20 genomic sequences were aligned to this gene, substantially lower numbers of the TN20 sequences mapped to the SCN P. dendritiformis-like gene (FIG. 2). Also, very few reads from TN20 matched a large intron spanning 4,000 to 6,000 bp, but the coding region of the gene was well covered (FIG. 2). The low TN20 read coverage suggested the SCN P. dendritiformis-like gene was reduced in copy number in the virulent TN20 SCN. A TaqMan assay was developed that compared the fold difference in copy number of the P. dendritiformis-like gene to a reference SCN gene HgFAR-1 (SEQ ID NO: 49). This assay was used to verify the copy-number reduction; an over 300-fold drop between TN10 and TN20 populations, of the SCN P. dendritiformis-like gene in the TN20 inbred SCN population (FIG. 3). The copy-number reduction also occurred in two other unrelated virulent SCN strains, (OP20 and OP50) but not in another non-virulent SCN strain (OP25), suggesting the copy number of this gene in the nematode population may be important for SCN virulence (FIG. 3).

SCN scaffold 1924 contained a previously identified bacterial-like biotin synthase gene (HgBioB) (Craig et al., J Nematol 41: 281-290, 2009). TN20 and TN10 Illumina genomic reads were aligned to the HgBioB gene to identify sequence polymorphisms. The HgBioB protein contained amino acid sequence differences between non-virulent TN10 and virulent TN20 SCN inbred lines at P24_A and R44_Q (see SEQ ID NOs: 44 and 45 for the nucleotide and amino acid sequences).

SCN scaffold 20, while larger than the others, did not contain an obvious HGT or SCN effector gene candidate.

SEQ ID NO: 44 is the BioB sequence; the two possible nucleotides at the two SNPs identified herein (position 70: C/G (TN10/TN20); position 132: G/A (TN10/TN20)) are italicized and underlined.

ATGCCACCCCCAATTGGCTCAATTATTTCCAAATGGACTTTCTCTGAGGC CCTTTCGGTGTTTTCACTC

CTTTCCCCGAACTCATTTTTCGTGCCC AAAATGTCCATCAGCAGCATCACAATCCAAGCC

AGTTCAAATCAGT ACGTTGTTGAGCATAAAAACGGGCGCGTGTCCGGAGAACTGTTCGTACTG TCCGCAGTCGGGCTACCATAAGACGGGGCTGAAGAAGGAGCCGTTGATGG AAGTGGAACAGGTGTTGGAAGCCGCTAAAAGAGCAAAGGCCAGCGGCGCG ACACGATTTTGTATGGGGGCGGCATGGAGGGGCCCGAAGGACCGCGACTT GGACAAAGTGTGCGAAATGGTGGCCAAAGTCAAACAATTGGGTGGCCTTG AAACATGCGCGACTTTGGGACTGCTCAAAAACGAGGGACAGGCGCAAAGA CTGAAGAAAGCGGGATTGGACTTTTACAACCACAACATCGACTGCTCCAA GGACTTTTACCACAAAATCATTACAACGCGCCGCTTTGATGACCGAATTT CGACCATTGAGAAAGTTCGTTCGGCCGGCATCAAAGTTTGCTGCGGAGGA ATTATCGGAATGGGAGAGAATAACGAAGAACGGGTGAAAATGCTCGTCAC ATTGGCCAATTTCGCACCTCCGCCCGAATCGGTGCCAATTAACAAATTAA TGCCCTTCCCCGGCACTCCGTTGGCCAATGCCCCGGCGCCCGACCCCTTC GATTTCGTGCGCACAATTGCCACGGCGCGTGTTCTGATGCCAATGGCTTA CATCCGACTGTCGGCTGGCAGAGAGCAAATGGCGGACGAATTGCAGGCAC TTTGTTTTTTAGCCGGTGCGAATTCACTTTTCTTTGGGGAAAAGTTACTA ACGGCGTCAAATCCAATGCCAGAAAAAGACAAAGAATTATTTCAACGATT GGGTCTCAAAAGAGAGCAAATTGAGGAGAAAAAAGCTGAACGGAATGACG AAAAAGTGACCTTGAACTTGTGA

Gene Structure and Transcript Variation:

The SCN P. dendritiformis-like gene in scaffold 385 was intriguing since it had the most dramatic difference between virulent and avirulent SCN in the allelic imbalance analysis, and appeared to be deleted or substantially altered in the virulent SCN parent. For this reason this sequence was chosen for further analysis. The SCN P. dendritiformis-like gene encodes a predicted protein of 326 amino acids (36.8 kDa) containing a 20-amino acid signal peptide and a 70 amino acid coiled-coil domain at amino acid positions 41 through 111. The coiled-coil region is similar to members of the target soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein (SNAP) receptor domain superfamily (t-SNARE, e8.0×10⁻³). SNARE proteins are highly conserved in eukaryotes and are involved in mediating membrane fusion events between cell membranes. The SNARE motif consists of a central polar amino acid residue (R or Q) flanked by hydrophobic amino acids in a heptad repeat pattern that interacts with other proteins in the SNARE complex and excludes water. The SCN P. dendritiformis-like protein contained these structural motifs when compared to known plant and animal t-SNARE proteins (FIG. 4), thus this sequence was named Heterodera glycines SNARE-like protein 1 (HgSLP-1; SEQ ID NO: 42). The central polar amino acid in HgSLP-1 was threonine, which is found in some t-SNARE-like proteins that alter eukaryotic membrane fusion, but is atypical for eukaryotic SNARE motifs (Paumet et al., PLoS ONE 4: e7375, 2009). Since soybean SCN-resistance genes encode proteins involved in membrane fusion (Cook et al., Science 338: 1206-1209, 2012; Matsye et al., Plant Mol Biol 80: 131-155, 2012), this nematode gene was further characterized.

The genomic sequence encoding the HgSLP-1 gene (SEQ ID NO: 40) contained 9 exons and 8 introns. However, the gene also showed evidence of intron sequence retention because most introns showed some coverage when Illumina cDNA reads were aligned to the genome sequence, with introns 3 and 8 showing the highest coverage (FIG. 5). If transcripts were produced containing intron sequences, the resulting proteins would be truncated due to stop codons in all of the intron sequences. The one exception is intron 3, which does not have a stop codon, but does have enough cDNA coverage so that one third of the transcripts could retain this intron. In this case, it would be expected that a protein 48 amino acids longer would be produced (FIG. 5). In addition, alignments of cDNA to the genomic sequence indicates a three-nucleotide deletion occurs 13% of the time due to an apparent alternative splice site at the beginning of exon 3. This alternative spliced form would produce a protein one amino acid shorter, missing Q107, which is at the end of the t-SNARE domain and thus could be functionally significant. Furthermore, the first exon of HgSLP-1 appears to be similar to PTR7 and Mer40 repetitive sequences identified in SCN expressed sequence tags (ESTs) B1748250 and CB824834, respectively, making the first exon, and related sequences more abundant than a single copy gene. Most of the ESTs that matched HgSLP-1 were only similar in the first repetitive exon, but two ESTs from SCN eggs (CB825264 and CA940412) were related to HgSLP-1, 73% identical over the first two exons, but only 57% identical overall. This suggests SCN expresses at least two forms of HgSLP, but the gene related to the EST is missing part of the t-SNARE domain, again suggesting it could have an altered function.

Silencing of HgSLP-1 by RNAi:

The deletion of the HgSLP-1 gene in virulent nematodes suggested that it might be possible to mimic this effect via the use of RNAi. To this end, non-virulent SCN containing HgSLP-1 were treated with four synthetic dsiRNAs directed against HgSLP-1. As a negative control, the same avirulent SCN were treated with the same amount of a non-nematode dsiRNA. Equal numbers of dsiRNA treated nematodes were inoculated onto susceptible and resistant (Rhg1 or Rhg4) soybean plants. After 30 days the number of cysts were counted. The number of cysts that formed on either type of resistant plant did not statistically differ between SCN populations treated with dsiRNAs that targeted the control gene or HgSLP-1. However, the number of cysts that grew on the susceptible plants was statistically higher for the plants inoculated with SCN treated with the dsiRNAs directed at the HgSLP-1 gene in comparison to the control (Table 3). RT-QPCR was conducted to detect the level of HgSLP-1 transcript from the dsiRNA treated J2s, but the expression of the gene in the J2 developmental stage was too low for reproducible quantification.

TABLE 3 Growth of RNAi treated SCN on nematode resistant and susceptible plants Cysts per plant Susceptible Rhg1 Rhg4 Plant HgSLP-1 NC HgSLP-1 NC HgSLP-1 NC 1  139^(a) 112 0 0 19 10 2 176 89 3 2 31 12 3 189 62 1 0 19 26 4 115 54 5 3 21 20 5 — 55 — 9 23 26 Mean ± 155 ± 74 ± 2.25 ± 2.8 ± 22.6 ± 18.8 ± S.E. 34 26 2.2 3.7 5 7.6 P     0.009 0.790 0.380 HgSLP-1 denotes SCN treated with dsiRNAs for HgSLP-1 and NC denotes SCN treated with negative control dsiRNA. The “—” indicates the plant died before cysts could form.

HgSLP-1 Protein Localization:

The presence of a potential signal peptide at the N-terminus of HgSLP-1 suggested it was a possible secreted protein. Immunolocalization experiments were conducted to localize the protein in the nematode while it was parasitizing the plant to determine if it was expressed in a nematode cell type that might secrete the protein from the nematode into the plant. To do this, peptide antibodies to HgSLP-1 were produced and incubated with nematode infested root sections. When the HgSLP-1 antibodies were detected via florescent microscopy, the antibodies bound to a subventral esophageal gland, indicated by the extensive florescent signal emitted from the basal cell body (FIG. 6A). Distinct antibody staining was also observed in the gland extension, metacorpus and esophageal lumen and stylet (FIGS. 6B and C). While florescent signals were also observed in plant cells walls adjacent to the nematode, we do not interpret this signal as the in planta location of HgSLP-1 since florescent signals are also observed in plant cell walls near the nematode in control sections. However, florescent signals in an esophageal gland or stylet are never observed in control sections. The fact that florescent signal is present in the esophageal lumen and stylet is consistent with the HgSLP-1 being secreted from the nematode since at this point the protein would have passed the valves in the metacorpus and there would be not further barriers to HgSLP-1 leaving the nematode. However, this data does not show that HgSLP-1 is injected into the nematode feeding cell. Future higher-resolution microscopic studies on SCN feeding cells will be required to address the plant subcellular location of HgSLP-1. Since HgSLP-1 appeared to be secreted, it is reasonable to assume that it could be injected into the nematode feeding cell and interact with soybean proteins.

Characterization of HgSLP-1 by Co-Expression/Co-Purification:

The coiled-coil domain of HgSLP-1 was similar to domains found in t-SNARE proteins, suggesting that a number of plant proteins involved in membrane fusion might bind this nematode effector. Because one of the genes at the Rhg1 locus is predicted to encode an α-SNAP, we hypothesized that the soybean Rhg1 α-SNAP might directly bind to HgSLP-1 because α-SNAPs and t-SNARE proteins interact during the membrane fusion cycle. To test this hypothesis, we placed the genes for HgSLP-1 and the soybean α-SNAP from the Rhg1 locus in an Escherichia coli dual expression vector. For HgSLP-1 two forms were used in two different constructs, a full length and a form lacking the signal peptide. Likewise, a full-length α-SNAP gene was used, however, a C-terminal six-histidine (6×his) tag was added to allow for purification of the expressed α-SNAP protein. The E. coli were induced to co-express both proteins and then the bacterial cells were lysed under non-denaturing conditions and the proteins purified. As a negative control we expressed just the HgSLP-1, which lacks the 6×his tag, in E. coli and attempted to purify it in parallel with the co-expressed proteins. Protein gel blots were conducted on the purified proteins and they were detected using both anti-HgSLP-1 and anti-soybean α-SNAP antibodies (FIG. 7). In the lanes containing proteins purified from the co-expressed E. coli, both HgSLP-1 and soybean α-SNAP could be detected within 20 minutes, suggesting they were both abundant in the purified proteins. Trypsin digestion and mass spectrometry also detected fragments of both proteins in purified samples. The negative control, the full size HgSLP-1 alone, did not purify, but the protein was easily detected in the initial total E. coli lysates and was equivalent in amount to the co-expressed HgSLP-1, which indicates it did not bind to the chromatography beads (FIG. 7). This co-purification of HgSLP-1 and soybean α-SNAP occurred even when the metal affinity chromatography beads were very stringently washed, suggesting that the two proteins bind to each other.

To gain additional evidence of protein-protein interaction, the total E. coli protein extracts described above containing HgSLP-1 or both HgSLP-1 and α-SNAP proteins were independently run over a gel filtration column. The fractions were collected and assayed for the presence of the HgSLP-1 protein via antibody dot blots. The HgSLP-1 (36.8 kDa) alone eluted at fraction 23 slightly sooner than the chymotrypsinogen A standard (25 kDa) which eluted at fraction 30, which is consistent with its expected size. However, when extracts containing both HgSLP-1 and α-SNAP were run through the column, the HgSLP-1 eluted at fraction 15, very close to the albumin standard (67 kDa) that eluted at fraction 12. The shift in elution of HgSLP-1 is consistent with this protein binding to α-SNAP and confirms the co-purification experiments described above. In both protein extracts, early fractions, particularly fraction 1, also contained HgSLP-1. Since fraction 1 contains proteins too large to be fractionated by the column matrix, it suggests a larger HgSLP-1: a-SNAP complex (dimer) may form (FIG. 8).

Discussion

The identification of the molecular mechanisms that plant parasitic nematodes use to evade or suppress host plant resistance is of great practical significance, since understanding this process could lead to broader and more durable resistant plants or to rapid diagnostic tests to predict the virulence profile of field nematode populations. A genetic approach to the identification of nematode virulence genes makes few assumptions about the underlying nature of the genes controlling the virulence phenotype. Past genetic studies on SCN virulence indicated one or two genes control the nematodes ability to reproduce on Rhg1 and Rhg4 resistant plants (Dong & Opperman, Genetics 146: 1311-1318, 19). This foundational study showed that SCN was a viable genetic system; however, it was also clear that SCN lacked a genetic infrastructure (a genome sequence and sequence polymorphisms) needed for map-based cloning of the genes controlling the virulence phenotype.

Part of the problem has been resolved with the development of the semi-quantitative GoldenGate SNP assays for SCN (Carlson et al., Human Molecular Genetics 15: 1931-1937, 2006) that can be used for genetic mapping and for map-based cloning via allelic imbalance (Wong et al., Nucleic Acids Res 32: e69, 2004) or bulk segregant analysis (Hyten et al., Crop Science 49: 265-271, 2009). In our study, three SNPs (212, 1035 and 1533) showed a consistent difference in SNP frequency when the bulk nematodes were grown on resistant and susceptible plants. Our criteria for identifying candidate virulence genes in the scaffolds of interest was based on the hypothesis that an SCN virulence gene might encode an effector protein or may have entered the genome via HGT from a microorganism and that the putative virulence gene should have a clear sequence polymorphism(s) between virulent and avirulent parents. Due to a lack of known SCN effector proteins in the SCN scaffolds under investigation, the putative HGT events in two of the scaffolds became the focus of our attention. The scaffold with SNP 1035 contained HgBioB, the scaffold with SNP 212 contained the HgSLP-1, and the third SNP-containing region is still under analysis.

HgBioB

Biotin functions as a carboxyl carrier for biotin dependent carboxylases, which are critical for fatty acid metabolism and amino acid catabolism. Biotin has also been shown to play a role in cell signaling, epigenetic regulation of genes, chromatin structure (Zempleni et al., Biotin. BioFactors 35: 36-46, 2009; Zempleni et al., The Journal of Nutrition 139: 2389-2392, 2009), and recently in microbial pathogenesis, making it a good candidate for a SCN virulence gene (Feng et al., Molecular Microbiology 91: 300-314, 2014).

In general, multicellular animals, including SCN, have lost the ability to synthesize biotin de novo. It is assumed the gene loss occurred because animals can simply acquire the vitamins through their diet. Thus, it seems unusual that SCN, an animal and a parasite, would express biotin synthase. The discovery of HgBioB in one of the scaffolds associated with SCN virulence was very significant, since this gene had previously been identified and speculated to be involved in SCN virulence (Craig et al., J Nematol 41: 281-290, 2009). In fact, the SCN SNP 1035 associated with SCN virulence was in the HgBioB gene, making it the best candidate virulence gene in the scaffold. HgBioB has been predicted to be functional, since it retains a conserved active site, but the virulent and avirulent SCN appear to have slightly different amino acid sequences. These amino acid sequence differences could alter biotin synthase enzymatic activity and thus could be the basis of this virulence trait. The exact role HgBioB could play in virulence is unclear, but we have previously speculated it could be a method for the nematode to circumvent SCN resistance, if part of the mechanism of resistance is caused by the plant reducing biotin availability during a nematode resistance response. It should be noted, that the nematode does not have the complete biotin biosynthetic pathway, but only the last enzyme in the pathway. So, if a plant reduced biotin synthesis at the last step, as a mechanism to starve the nematode parasite, the precursors to biotin may still be available for conversion to biotin via HgBioB (Craig et al., Mol Biol Evol 25: 2085-2098, 2008; Craig et al., J Nematol 41: 281-290, 2009). In this scenario a more enzymatically active biotin synthase enzyme, in the virulent SCN, could give the nematode a competitive advantage. It would be necessary to measure the levels of biotin in SCN feeding cells of susceptible and resistant soybean plants to test this hypothesis.

HgSLP-1

The region of the SCN genome that contains SNP 212 encodes HgSLP-1, a gene that appears to have entered the SCN genome via HGT. This gene is ˜6,000 bp from SNP 212 and thus might be expected to provide a strong selection on the SNP in the allelic imbalance experiments. The SCN gene encoding HgSLP-1 is homologous to a bacterial protein from P. dendritiformis that has no known function, but was predicted to contain a viral hemagglutinin stalk and Sec3_C domains. The area of homology between the two proteins is in the viral hemagglutinin stalk domain which is structurally related to the SNARE motif found in HgSLP-1 in that they both, form coiled-coil domains and are involved in protein-protein interactions that occur during membrane fusion events in the cell (Skehel & Wiley, Cell 95: 871-874, 199).

The presence of SNARE motifs in bacterial proteins is not unusual, in fact such proteins act as virulence effectors in intracellular human pathogens such as Chlamydia, Mycobacterium, Salmonella and Legionella (Wesolowski & Paumet, Virulence 1: 319-324, 2010). In these intracellular bacteria the SNARE domain acts as a mimic and suppresses host membrane fusion events which aids the bacteria by preventing the phagosome membranes from fusing with lysosomes and killing the bacteria. The bacterial SNARE-like proteins directly bind to host v-SNAREs to prevent membrane fusion (Delevoye et al., PLoS Pathogens 4: e1000022, 2008). This type of SNARE-like protein that suppresses membrane fusion could also play a similar role in SCN parasitism of susceptible soybean.

While little direct research has been conducted on this topic in plant-nematode interactions, transmission electron microscopy images of cyst nematode feeding cells consistently showed endoplasmic reticulum (ER) membrane association with nematode feeding tubes (Sobczak et al., Nematology 1: 363-374, 1999) and enlarged ER membranes in syncytia (Endo, “Cellular responses to infection.” In: Riggs & Wrather, editors. Biology and management of the soybean cyst nematode. St. Paul, Minn.: American Phytopathological Society. pp. 37-49, 1992; Sobczak & Golinowski, “Cyst Nematodes and Syncytia.” In: Jones et al., editors. Genomics and Molecular Genetics of Plant-Nematode Interactions. Dordrecht: Springer. pp. 61-82, 2011), suggesting alteration of plant membranes might be essential for nematode feeding. However, such images do not shed light on whether nematodes suppress membrane fusion during feeding cell formation. In addition, the formation of plant cell plate is mediated by SNARE proteins; therefore, it is not unreasonable to suggest that the formation of the syncytia via the breakdown of neighboring cell walls may also be impacted by HgSLP-1 if it were able to interact with SNARE proteins which control this process (Zhang et al., PLoS ONE 6: e26129, 2011). Transcriptome data from syncytia suggests that some alteration of host secretion machinery occurs since soybean α-SNAP is not abundantly expressed in susceptible plants (Matsye et al., Plant Mol Biol 80: 131-155, 2012; Matsye et al., Plant Mol Biol 77: 513-528, 2011).

The role of plant membrane fusion in SCN resistant plants is more firmly established since one of the Rhg1 resistance genes encodes a α-SNAP protein. These α-SNAP proteins may confer SCN resistance by themselves (Matsye et al., Plant Mol Biol 80: 131-155, 2012; Pant et al., Plant Mol Biol., 85:101-121, 2014) or in combination with the other resistance genes at the Rhg1 locus (Cook et al., Science 338: 1206-1209, 2012). The Rhg-1 α-SNAP proteins show significant polymorphisms between and within SCN resistant and susceptible soybean lines (Cook et al., Plant Physiol, 2014). The α-SNAPs are part of the complex of proteins that regulate membrane fusion and are highly conserved among eukaryotic organisms (Ferro-Novick & Jahn. Nature 370: 191-193, 1994), but it has been suggested that the Rhg1 α-SNAPs are specialized for conferring SCN resistance (Cook et al., Science 338: 1206-1209, 2012; Cook et al., Plant Physiol, 2014). To initiate membrane fusion between a vesicle and a target membrane, a complex of proteins must interact; a vesicle will have a membrane bound v-SNARE (also referred to as R-SAREs, Vamp or synaptobrevin proteins), while the target membrane will have a different membrane bound t-SNARE protein (also referred to as Q-SAREs or syntaxin proteins) (Fasshauer et al., Proc Natl Acad Sci USA 95: 15781-15786, 1998). The complex also contains a protein called SNAP-25 that binds to the other SNAREs to form a stable trans-SNARE complex where coiled-coil domains bind these proteins together (Fasshauer et al., Proc Natl Acad Sci USA 95: 15781-15786, 1998; Chen & Scheller, Nature Reviews MCB 2: 98-106, 2001) (FIG. 9A). The soluble protein α-SNAP is an adapter protein and binds to the t-SNARE. NSF and to a lesser extent SNAP-25 (Hayashi et al., The EMBO J 14: 2317-2325, 1995) and stimulates the NSF ATPase to disassemble the cis-SNARE complex after membrane fusion (Vivona et al., J Biol Chem 288: 24984-24991, 2013). α-SNAP proteins bind to t-SNAREs via coiled-coil domains both in trans-SNARE complexes and independently (Barnard et al., Molecular Biology of the Cell 7: 693-701, 1996). When α-SNAP binds to free t-SNAREs in a cell, this interaction blocks the protein and prevents membrane fusion events (Rodriguez et al., PLoS ONE 6: e21925, 2011). Thus, it seemed possible that if the HgSLP-1 could act as a t-SNARE mimic and bind to an α-SNAP, or a soybean v-SNARE, it might prevent membrane fusion events in the nematodes' feeding cell by sequestering these required plant membrane fusion proteins (FIG. 99).

This model is supported by the observations that the HgSLP-1 protein is expressed and secreted from an esophageal gland cell while the nematode is parasitizing the plant and the observation that HgSLP-1. We should note that while our data does suggest HgSLP-1 is secreted from a subvental gland, due to its presence in the esophageal lumen and stylet, our immunolocalization data in not clear in showing the protein entering the feeding cells. While it is likely that HgSLP-1 enters the nematode feeding cell, future higher-resolution immunolocalization experiments will need to be conducted to resolve the in planta localization of HgSLP-1. The model is also supported by the data that indicates HgSLP-1 and soybean α-SNAP bind to each other when co-expressed and co-purified from E. coli. The consequence of such an interaction of HgSLP-1 and α-SNAP might be to suppress membrane fusion events and this might disrupt both plant defense pathways and numerous normal cellular activities.

Exactly how HgSLP-1 might mitigate host defenses in soybean lacking the Rhg-1 loci is unclear, but it has been demonstrated that over expression of α-SNAP can induce syntaxin 31 in soybean and that this t-SNARE protein enhances SCN resistance (Pant et al., Plant Mol Biol., 85:101-121, 2014), thus blocking this interaction may promote susceptibility. It has also been demonstrated in tobacco (Nicotiana tabacum) that by blocking syntaxin SYP132, a t-SNARE protein that transports vesicles containing PR proteins, increased bacterial susceptibility results (Kalde et al., Proc Natl Acad Sci USA 104: 11850-11855, 2007). Similarly, resistance to Peronospora parasitica is mediated via VAMP721/722 vesicles in Arabidopsis (Kim et al., The Plant J: CMB 79: 835-847, 2014), suggesting vesicle transport is a critical component for host-plant resistance to several plant pathogens. Thus, it seems plausible that HgSLP-1 could block the vesicle-mediated transport of anti-nematode proteins or metabolites in a similar way and inactive this type of basic host defense mechanism. However in Rhg1-mediated resistance we have a different scenario where up to 10 copies of Rhg1 α-SNAP and associated resistance genes. In effect, the α-SNAP in the resistant plant could be specialized to bind the nematode SNARE mimic and block its effect (FIG. 9C). This idea that the Rhg1 α-SNAP is specialized for nematode resistance was suggested in the original paper describing the Rhg1 loci (Cook et al., Science 338: 1206-1209, 2012).

However, our data suggests the role of HgSLP-1 and its related family members in nematode virulence or pathogenicity is not simple. Different isolates of SCN populations had variable copies of the HgSLP-1 gene and the RNA-seq data suggests that the mRNA encoded by the gene is alternatively spliced and forms containing intron sequences may be produced. This diversity of SCN SNARE like proteins could reflect the nematodes attempt to adapt to different soybean cultivars with different complements of α-SNAP proteins or other binding partners of HgSLP-1. Indeed, the apparent reduction in copy number of HgSLP-1 from TN20 and other nematode strains may reflect a resistance evasion mechanism used by the nematode as several highly virulent SCN strains appear to have reduced numbers of the gene in the population of nematodes. If HgSLP-1 is important for establishing a feeding cell, one might expect that the lack of this protein may impair the growth and development of virulent SCN lacking the gene. However, we suggest that virulent nematodes may express different forms of SCN SNARE-like protein (such as the ESTs CB825264 and CA940412) that are not recognized by the Rhg1 α-SNAPs, due to mutations in the SNARE domain, and thus may compensate for the loss of HgSLP-1 expression (FIG. 9D). While the lack of HgSLP-1 may play a role in evading host plant defense mechanisms, other potential virulence genes, such as the HgBioB or the gene near SNP 1533 are probably also required for the virulence phenotype.

Some of the SCN strains we tested for HgSLP-1 were the genetically characterized inbred SCN strains OP20, OP25 and OP50 (Dong & Opperman, Genetics 146: 1311-1318, 1997). The genetic study of the OP SCN inbred strains established that SCN virulence was controlled by one or two genes, depending on the type of resistant plants and nematode strains were tested. This landmark study established that virulence on Rhg1-type resistance was genetically controlled by the dominant Ror1 gene (reproduction on resistant) in the virulent strain OP50, but OP50 growth on Rhg4/Rhg1-based resistance was controlled by the recessive ror2 gene. The virulent inbred SCN strain OP20 was shown to require two virulence genes to reproduce on the Rhg1 source of resistance. Dong & Opperman (1997) suggested that SCN utilizes several mechanisms to overcome Rhg1-based resistance, but only one gene, ror2, to overcome Rhg1/Rhg4-type resistance. The avirulent SCN strain OP25 was unable to reproduce on any source of resistance. When these nematodes were tested for the presence or absence of the HgSLP-1, the gene was present in OP25 (avirulent SCN), but nearly absent or reduced in copy number in OP20 and OP50 (both virulent on both Rhg1 and Rhg1/Rhg4-type resistance). It is interesting to note, that even though OP20, OP50 and TN20 are not related to each other (two are from North Carolina and the other from Missouri) the nematode strains have a similar virulence profile in that they grow on most SCN resistant plants. More work is needed to confirm the correlation between lower copy number of HgSLP-1 and broad SCN virulence. However, it is interesting that the ror2 virulence gene that controls growth on the Rhg1/Rhg4-type resistance is a recessive gene. The deleted HgSLP-1 gene in most of the nematodes in this strain would be expected to act as a recessive gene, thus it may be that ror2 is in fact this deletion or lack of HgSLP-1 or that the deletion of SCN virulence genes to evade host plant resistance is a common mechanism.

Overall, HgSLP-1 could play a role in SCN virulence, via its absence, but it could be involved with feeding cell formation if alterations of membrane fusion would be beneficial in this process. It seems plausible that HgSLP-1 may have other binding partners in the plant cell in addition to the Rhg1 α-SNAP. It will be interesting in the future to test other α-SNAP variants to determine their binding kinetics to HgSLP-1 in its various forms. It will also be important to follow up on the involvement in biotin in Rhg1 mediated resistance by further analysis of the biotin biosynthetic enzymes in the plant and nematode. The virulence gene identification approach described in this paper should be adaptable to other plant-nematode systems to associate SNPs with nematode phenotypes of interest. In addition, new high throughput methods of DNA sequencing and genotyping will lower the cost and increase the resolution of this type of genetic screening approach making it applicable to any sexually reproducing nematode species. Increasing our knowledge of how nematodes evade/suppress host plant resistance may lead to more durable SCN resistant plants or to improved methods of monitoring virulent nematode populations, which in turn will aid in the management of these damaging pathogens.

Example 2 Quantification of HgSLP-1 Copy Number to Assess Virulence in a Nematode Population

This Example describes a study of nematode virulence, employing quantification of HgSLP-1 copy number as a measure of virulence in a field population.

Field populations of SCN taken from 14 different samples were grown in the lab on susceptible soybean Essex. The nematodes were allowed to increase for about 6 weeks, then the cysts were extracted and popped to release the eggs. Some of the eggs were used to inoculate susceptible (Essex) and resistant (PI88788) soybean (equal numbers of eggs were used in the inoculations).

The rest of the eggs were used to extract DNA. The eggs were added to 2 ml plastic tubes, excess water removed, and then two 3.2 mm diameter stainless steel beads were added. The prepared sample tubes were frozen in liquid nitrogen and then shaken at high velocity on a mini-beadbeater-16 model 607 (Biospec Products) cell disrupter. The frozen pulverized eggs were transferred back to liquid nitrogen for storage until the DNA was extracted using a DNAeasy Blood and Tissue kit (Qiagen) following manufacturer's instructions. The copy number assay for HgSLP-1 was performed as described above.

In this analysis, we found that some of the nematodes did not grow (this could be since they picked up a disease from the field, a common problem), thus not all the data points have a growth test. The ones that did, appeared to support the theory that low copy numbers of HgSLP-1 allow the nematode to grow on resistant plant PI88788.

Results are shown in FIG. 10. The QPCR assay measures the frequency of HgSLP-1 (copy number varies) in each nematode population relative to HgFAR-1 (copy number does not vary). The smaller/shorter the bars in FIG. 1 are SCN populations predicted to be the more virulent. Samples 1, 7, 9, 10 SCN were able to grow on susceptible and resistant soybean plants (PI88788) and have relatively low copies of HgSLP-1. Sample 5 SCN were also able to grow on PI88788 SCN, but had intermediate HgSLP-1 copy number. However, Sample 6 SCN, which has a relatively higher copy number of HgSLP-1, did not grow on PI88788, but did grow on a susceptible soybean plant. The other sample SCN populations did not grow on either susceptible or resistant soybean, thus their virulence level is unknown.

Example 3 Detection/Quantification of SCN Virulence in Soil

This Example provides an exemplary system for detecting the resistance-breaking tendency (virulence) of nematodes (e.g., SCN) in a field/soil area.

Nematodes (e.g., cysts or eggs) are isolated from a soil sample taken, for instance, from a field in which soybean is or may in the future be planted. By way of example, the soil sample may be an aggregated sample, taken from multiple sub-locations within a target area that are then mixed to obtain a sample more representative of a possibly heterogeneous distribution over the source location area. Nematode, egg, and cyst isolation techniques are well known to those of skill in the art. DNA is then obtained from the isolated nematode population(s), for instance using art-recognized techniques. Representative techniques are provided herein.

In one embodiment the copy number of the HgSLP-1 gene is determined. Usually, the copy number is determined relative to a gene of stable/largely non-variant copy number, and the copy number ratio is used as in Example 1 and 2 above. The lower the copy number of HgSLP-1 in the tested nematode population, the more likely the population is to be virulent (that is, capable of growth on otherwise resistant soybean plants).

In another embodiment, the sequence of the BioB gene is determined in the population of nematodes, specifically to determine the sequence at positions 70 and/or 132 (numbering as in SEQ ID NO: 44). BioB sequence containing a C at position 70, and/or a G at position 132, is expected to be from a non-virulent nematode; while BioB sequence containing a G at position 70, and/or an A at position 132, is expected to be from a virulent nematode. In a mixed population, the relative amount of each sequence can be calculated to determine a relative level of virulence in the population.

The results of such a virulence analysis can be used, for instance, in decisions regarding how to utilize or treat the field from which the soil sample(s) were obtained. By way of example, in heavily virulent fields (that is, fields with a relatively high level of virulent nematodes), the field might be aggressively treated to eradicate the SCN. Alternatively, such a field might be turned, for instance for a number of seasons or years, to a use other than growing soybean—that is, rotated to a different use—or rotated to a soybean cultivar having a different source of resistance than that found in PI88788 (e.g., distinct from the α-SNAP-mediate resistance described herein) (see, e.g., Luedders & Dropkin, Crop Sci. 23, 263-264, 1983).

This disclosure provides systems for analyzing nematode (e.g., soybean cyst nematode) genetics and identifying resistance/virulence and other genes, and the identification of two putative nematode virulence genes. The disclosure further provides methods of using the presence, absence, and/or sequence of these genes to detect virulent nematode populations, for instance in soil. It will be apparent that the precise details of the methods described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below. 

The invention claimed is:
 1. A method, comprising: isolating soybean cyst nematodes (SCN) from at least one soil sample from a source location; obtaining DNA from the isolated nematodes; measuring the relative copy number of the Heterodera glycines SNARE-like protein-1 gene (HgSLP-1) gene comprising the nucleic acid sequence of SEQ ID NO: 40 or SEQ ID NO: 41 in the DNA obtained from the isolated nematodes; detecting a low relative copy number of HgSLP-1, wherein a low relative copy number of HgSLP-1 is indicative of SCN virulence in the soil sample; and planting in soil at the source location a soybean cultivar having a different source of resistance than that in resistant cultivar PI88788.
 2. The method of claim 1, wherein measuring the relative copy number of the HgSLP-1 gene in the nematode DNA comprises measuring the abundance of the HgSLP-1 sequence compared to the copy number of a control nematode gene using quantitative PCR.
 3. The method of claim 2, wherein the control nematode gene is Heterodera glycines fatty acid and retinol binding protein-1 gene (HgFAR-1).
 4. The method of claim 2, further comprising preparing a ratio of the copy number of HgSLP-1 to the copy number of the control nematode gene, and wherein a ratio of ≤0.3 indicates SCN virulence in the soil.
 5. The method of claim 2, wherein the quantitative PCR uses primer pair SEQ ID NOs: 18 and 19 or NOs: 46 and 47 to amplify HgSLP-1 DNA.
 6. The method of claim 1, wherein measuring the copy number of the HgSLP-1 gene in the nematode DNA comprises detecting HgSLP-1 DNA using a labeled probe having the sequence of SEQ ID NO: 20 or
 48. 7. The method of claim 3, wherein the quantitative PCR uses primer pair SEQ ID NOs: 15 and 16 to amplify HgFAR-1 DNA.
 8. The method of claim 3, wherein measuring the copy number of the HgFAR-1 gene in the nematode DNA comprises detecting the HgFAR-1 DNA using a labeled probe having the sequence of SEQ ID NO:
 17. 9. A method, comprising: isolating soybean cyst nematodes (SCN) from at least one soil sample from a source location; obtaining DNA from the isolated nematodes; detecting the sequence of the Heterodera glycines bacterial-like biosynthase gene (HgBioB) gene in the DNA obtained from the isolated nematodes; detecting presence of C at position 70 and A at position 132 (numbered with reference to SEQ ID NO: 44) within HgBioB, wherein the presence of C at position 70 and A at position 132 (numbered with reference to SEQ ID NO: 44) within HgBioB is indicative of SCN virulence in the soil sample; and planting in soil at the source location a soybean cultivar having a different source of resistance than that in resistant cultivar PI88788. 